一种与聚羟基脂肪酸酯生物合成相关的新型水合酶结构生物学研究
|
摘要
辣椒疫霉是一种世界性土传病害，辣椒疫病严重影响世界各国的辣椒生产，造成了巨大的经济损失。该病寄主范围广，可以侵染辣椒、西葫芦、黄瓜、茄子、番茄等20多种作物。近年来有关其发病机制、致病机理、防治策略等相关报道层出不穷，而从类似的致病菌中寻找有益基因为人类所利用却鲜有报道。
2008年辣椒疫霉全基因组序列草图由美国能源部基因研究所公布于众（网址http://genome.jgi-psf.org/Phyca11/Phyca11.home.html）并不断更新完善。通过生物信息学分析，越来越多的人们感兴趣的基因得以被发现进而为揭示其相关功能并被人们所利用提供了便利。我们研究的类单胺氧化酶C类水合酶（MaoC-like hydratase，MaoC）即是在对其全基因组序列分析的基础上获得的一个新基因。MaoC在连接脂肪酸的β-氧化到聚羟基脂肪酸酯（Polyhydroxyalkanoate，PHA）的生物合成过程中具有加水作用，是最近被发现的一种新型R型水合酶。
全球化的环境问题使得人们越来越关注于可生物降解材料的开发与使用。在被研究中的可生物降解材料中，PHA以其所具有的生物降解性、生物相容性、热塑性和机械性能成为一种理想的“绿色塑料”替代物。所以，许多科学家正在尝试研制低成本高产出的PHA生产方案。PHA生产成本的降低可以使其成为那些在自然坏境中很难分解的传统塑料的理想替代物，即作为“绿色塑料”被使用。MaoC即是首先在大肠杆菌中被发现的一种新型R型水合酶，它在脂肪酸的β-氧化到PHA的生物合成过程中起重要作用：MaoC作为R型水合酶的新成员可以提供PHA合成的前体物质，R-3-酮酰基辅酶A。
我们在辣椒疫霉菌中克隆到了一个新MaoC基因，获得了其蛋白晶体并解析了其高分辨率三维结构，以结构为基础对其功能进行了深入研究。具体总结如下：
1.在致病菌辣椒疫霉中克隆到一个有益基因MaoC，对其进行了原核表达，并对表达出的重组蛋白利用亲和层析、阴离子柱交换层析、分子排阻层析等手段进行了纯化，得到了具有较高纯度的稳定重组蛋白；利用特异性的酶活实验以巴豆酰辅酶A为底物对重组纯蛋白进行了酶活测定，结果显示重组纯蛋白具有乙酰辅酶A水合酶活性，达到58.1U/mg，MaoC因其乙酰辅酶A水合酶活性而成为与PHA合成相关的众多酶类的一员，是一种新型的R型水合酶，为PHA的合成提供前体物质，R-3-酮酰基辅酶A；
2.采用气相扩散悬滴结晶法获得了MaoC高质量蛋白晶体，并用分子置换法解析出分辨率达1.93的晶体结构；对其三维结构进行了细致描述与分析：MaoC具有R型水合酶类典型的结构折叠，分为明显的N-区域和C-区域，两个区域间由一个盐桥相连；具有典型的称为“热狗折叠”的保守结构域，并且N-区域不完整，而C-区域是完整的“热狗折叠”；MaoC通过四个α螺旋相互作用形成了同源二聚体结构，且参与二聚化作用的氨基酸在R型水合酶中是高度保守的；结构上的精细分析与比对还发现MaoC存在一段潜在的活性抑制片段；
3.以序列和结构比对为基础，确定了MaoC的催化中心为以下三个氨基酸：D194、H199和G217。为了验证其催化作用我们做了三个定点突变：D194E、H199Q和G217A；对三个突变体重组蛋白进行了纯化，并对纯蛋白进行了圆二色性、蛋白杂交和分子排阻层析验证，以确定其分子量及蛋白的真实性等；酶活测定结果显示三个氨基酸中D194和H199对MaoC行使酶活性起重要作用，G217起次要作用，具体作用方式为：D194通过吸引催化水分子上的一个质子而激活水分子，活化了的水分子攻击底物巴豆酰辅酶A上的碳原子，同时H199对底物上的碳原子贡献一个质子以使反应顺利进行，而G217通过氢键作用与底物硫酯键上的碳酰基结合，起到稳定底物的作用；
4.二聚体是MaoC的活性形式，MaoC通过四个α螺旋相互作用形成了同源二聚体结构。通过序列和结构比对分析发现，参与二聚化作用的氨基酸在不同的R型水合酶中是高度保守的，同时大部分已报道的R型水合酶结构均为二聚体结构，并且与MaoC具有类似的作用方式。基于结构上的分析与比对，我们推测二聚化作用对MaoC行使酶活性是必须的。为了验证这一推测，我们做了三个定点突变：R23D，M27E和L191D；对三个突变体纯蛋白进行分子排阻层析验证表明三个突变体均成功破坏了MaoC的二聚化作用，成为单体蛋白；酶活测定结果显示三个突变体单体蛋白均丧失活性，证实二聚化作用对MaoC行使酶活性是必须的；本研究首次通过生化实验证实二聚化作用对MaoC酶活起关键作用，考虑到二聚化作用在R型水合酶类蛋白上的普遍存在性，我们的结论即二聚化作用对其行使正常酶活是必须的可能对此类蛋白具有普遍适用性；一个单体结构可能会更好的解释其机制，然而我们对MaoC单体蛋白的结晶实验未能成功；
5.以结构为基础我们对MaoC进行了酶工程改造，以期得到与野生型蛋白相比具有较高酶活的突变体。结构分析表明一段非保守的插入片段可能对MaoC的酶活调节起作用。如果我们的假设正确的话，删除此片段将会对MaoC的活性产生影响。基于此假设我们对此区域进行了定点突变与片段删除突变，并对纯化后的突变体重组蛋白进行了酶活测定，结果显示两个突变体分别有较大幅度的活性提高：突变体Del63-71酶活为91.2U/mg，约为野生型蛋白的1.5倍；突变体Del63-88酶活为126.7U/mg，约为野生型蛋白的两倍；而定点突变对MaoC酶活基本无影响；分子排阻层析实验表明突变体活性的增强与其聚合状态无关，因为两个活性增强突变体均为二聚体；我们的研究表明MaoC上非保守的插入片段对其活性有抑制作用。
随着世界环境问题变得越来越严峻，发展绿色环保塑料以改善环境迫在眉睫。在本研究中，我们报道了一个与PHA生物合成相关的新型水合酶结构，并且对其活性调节机制进行了研究与改进，这些研究将为以更低的成本生产PHA提供有效信息。
Phytophthora capsici is a worldwide soil borne disease. Pepper Phytophthora blight hasserious impact on pepper production all over the world, and it causes huge economic losses. Ithas a broad host range including pepper, pumpkin, cucumber, eggplant, tomato, and so on, upto more than20kinds of plants. In recent years there are many reports on its pathogenesis,pathogenic mechanism and control strategy, while there are few reports on searching for somebeneficial genes to serve mankind from such pathogens.
In2008, the whole-genome draft sequence data from Photophthora capsici was madeavailable and updated constantly on line(http://genome.jgi-psf.org/Phyca11/Phyca11.home.html) by U.S. Department of Energy JointGenome Institute-DOE JGI. By bioinformatic analysis, more and more interesting genes arebeing found and this benefits us to reveal its function and be used by mankind. In our study,MaoC-like hydratase (to be shorten for MaoC), is one of such genes based on analysis of thegenome sequences. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved inlinking the β-oxidation and the polyhydroxyalkanoates (PHAs) biosynthetic pathways, hasbeen identified recently.
Problems concerning with the global environment have arisen much interest in thedevelopment of biodegradable polymers. Among the several biodegradable polymers underresearch, PHAs are good candidates for “green plastic” due to their biodegradable,biocompatible, thermoplastic, and mechanical properties. Because of this, considerableresearch has been undertaken to develop low cost and high efficiency processes for theproduction of PHAs. Reducing the cost of PHAs production may make PHA competitive as a“green plastic” alternative to conventional plastics which are difficult to degrade underrandom conditions. MaoC, possessing activity of (R)-hydratase involved in linking theβ-oxidation and the PHA biosynthetic pathways, has been recently identified in the fadBmutant Escherichia coli strain. MaoC is a new (R)-hydratase that catalyzes the (R)-specifichydration of the β-oxidation intermediate2-trans-enoyl-CoA to (R)-3-hydroxyacyl-CoA. ThusMaoC is an important enzyme in the biosynthesis of PHA because it supplies monomers thatare subsequently polymerized to form PHA by PHA synthase.
We cloned a new MaoC gene from Photophthora capsici, and the crystal structure of theenzyme has been resolved at high resolution. The detailed function has been clarified on thebasis of the structure, showing as follows:
1. We cloned a new beneficial gene named MaoC from pathogen Phytophthora capsici. MaoC was over-expressed in Escherichia coli and the recombinant MaoC was purified usingaffinity chromatography, anion-exchange chromatography and gel filtration chromatography.The purified recombinant MaoC was used to determine the Enoyl-CoA hydratase activityusing Crotonyl-CoA as the substrate, and the purified tagged MaoC showed the Enoyl-CoAhydratase activity of58.1U/mg towards Crotonyl-CoA. MaoC is a new member of(R)-hydratases which have been involved in biosynthesis of PHA, functioning as catalyzingthe (R)-specific hydration of the β-oxidation intermediate2-trans-enoyl-CoA to(R)-3-hydroxyacyl-CoA;
2. MaoC was crystallized using the hanging drop vapour diffusion method, and thecrystal structure of the enzyme was resolved at1.93resolution by molecular replacement.The detailed description and analysis of three-dimensional structure were carried out: Thestructure shows that MaoC has a canonical (R)-hydratase folding with an N-domain and aC-domain, located between them is the intervening bridge region. The crystal structure of theenzyme belongs to the so-called “hot dog” fold superfamily, compared to the C-domain, theN-domain has an incomplete hot dog fold. MaoC forms a homodimer that is mediated by fourshort α-helices from each monomeric MaoC, and the residues participating in dimerization arehighly conserved among the (R)-hydratases. Further analysis of the structure reveals that thereis a hypothetical inhibitory segment in regulating the activity of MaoC.
3. The active sites of MaoC (Asp-194, His-199and Gly-217) were identified by sequenceand structure alignment. To examine the importance of Asp-194, His-199and Gly-217forcatalysis, three mutants, D194N, H199Q, and G217A, were prepared and purified. To confirmthe presence and the apparent molecular mass of the purified mutant enzymes, circulardichroism, western blotting and gel filtration chromatography were carried out. The results ofactivity assay of the purified mutants show that Asp-194and His-199play a crucial role incatalysis, while Gly-217may be less important for catalysis. We propose that the threeresidues mentioned above play respective roles in the catalytic reaction of the enzyme asfollows. Asp-194may activate a water molecule by abstracting a proton from a watermolecule. The activated water molecule then attacks the carbon atom of crotonyl-CoA. Inaddition, His-199perhaps cooperatively donates a proton to the carbon atom of the substrate.Gly-217may hydrogen-bond to the carbonyl group of the thioester bond of the substrate.
4. In its structure, MaoC forms a homodimer that is mediated by four short α-helices fromeach monomeric MaoC. Based on the sequence and structure alignment, the residuesparticipating in dimerization are highly conserved among the (R)-hydratases, and most of thecrystal structures of hydratases resolved thus far are dimers with a similar interface to that in MaoC. These structural observations suggest that the dimeric MaoC is important for itsfunction. Supporting this conclusion, three mutations (R23D, M27E and L191D) disruptingthe dimerization of MaoC resulted in monomeric MaoC proteins in solution as indicated by agel filtration assay. More importantly, the enzymatic activities were completely absent in theMaoC mutant proteins. Together, these data strongly support the notion that MaoC must bedimerized to function. Our study provides the first biochemical evidence for the functionalsignificance of the dimer. A crystal structure of the monomeric MaoC will provide significantinsight into the mechanism underlying the requirement of MaoC dimerization for itsenzymatic activity. Unfortunately, all the efforts to crystallize a monomeric MaoC proteinfailed.
5. Based on the crystal structure, we made several mutants in engineering MaoC enzymeactivity in order to obtain mutants with higher activity compared to native MaoC. Structuralobservation suggests that a non-conserved insertion may have a role in regulating theenzymatic activity of MaoC. If this is the case, deletion of the insertion would generate aneffect on MaoC’s activity. To examine this possibility, we made single and deletion mutationssurrounding the hypothetical inhibitory segment and then assayed their effect on MaoC’sactivity. In full support of our prediction, one of the deletion mutant proteins, Del63-71,exhibited an activity of91.2U/mg in hydrolyzing enoyl-CoA, about1.5times that of the wildtype protein. Further deletion of the amino acids around the insertion (Del63-88) produced amore striking effect on promoting MaoC’s activity (126.7U/mg). In contrast, single mutationsin the insertion generated no significant effect on the activity of MaoC. The increased activityof the deletion mutant proteins was not caused by perturbation of the oligomerization status ofMaoC, because they both were eluted at a similar position to the wild type protein insize-exclusion chromatography. Collectively, our results indicate that an insertion region ofMaoC plays a role in inhibiting its activity.
As the environmental problem is becoming more and more serious all over the world, it isobviously significant to develop the “green plastic” to improve the environment. Here wereport the crystal structure of a new enzyme involved in PHA biosynthesis. The data in ourstudy reveal the regulatory mechanism of an (R)-hydratase, providing information on enzymeengineering to produce low cost PHAs.
2008年辣椒疫霉全基因组序列草图由美国能源部基因研究所公布于众（网址http://genome.jgi-psf.org/Phyca11/Phyca11.home.html）并不断更新完善。通过生物信息学分析，越来越多的人们感兴趣的基因得以被发现进而为揭示其相关功能并被人们所利用提供了便利。我们研究的类单胺氧化酶C类水合酶（MaoC-like hydratase，MaoC）即是在对其全基因组序列分析的基础上获得的一个新基因。MaoC在连接脂肪酸的β-氧化到聚羟基脂肪酸酯（Polyhydroxyalkanoate，PHA）的生物合成过程中具有加水作用，是最近被发现的一种新型R型水合酶。
全球化的环境问题使得人们越来越关注于可生物降解材料的开发与使用。在被研究中的可生物降解材料中，PHA以其所具有的生物降解性、生物相容性、热塑性和机械性能成为一种理想的“绿色塑料”替代物。所以，许多科学家正在尝试研制低成本高产出的PHA生产方案。PHA生产成本的降低可以使其成为那些在自然坏境中很难分解的传统塑料的理想替代物，即作为“绿色塑料”被使用。MaoC即是首先在大肠杆菌中被发现的一种新型R型水合酶，它在脂肪酸的β-氧化到PHA的生物合成过程中起重要作用：MaoC作为R型水合酶的新成员可以提供PHA合成的前体物质，R-3-酮酰基辅酶A。
我们在辣椒疫霉菌中克隆到了一个新MaoC基因，获得了其蛋白晶体并解析了其高分辨率三维结构，以结构为基础对其功能进行了深入研究。具体总结如下：
1.在致病菌辣椒疫霉中克隆到一个有益基因MaoC，对其进行了原核表达，并对表达出的重组蛋白利用亲和层析、阴离子柱交换层析、分子排阻层析等手段进行了纯化，得到了具有较高纯度的稳定重组蛋白；利用特异性的酶活实验以巴豆酰辅酶A为底物对重组纯蛋白进行了酶活测定，结果显示重组纯蛋白具有乙酰辅酶A水合酶活性，达到58.1U/mg，MaoC因其乙酰辅酶A水合酶活性而成为与PHA合成相关的众多酶类的一员，是一种新型的R型水合酶，为PHA的合成提供前体物质，R-3-酮酰基辅酶A；
2.采用气相扩散悬滴结晶法获得了MaoC高质量蛋白晶体，并用分子置换法解析出分辨率达1.93的晶体结构；对其三维结构进行了细致描述与分析：MaoC具有R型水合酶类典型的结构折叠，分为明显的N-区域和C-区域，两个区域间由一个盐桥相连；具有典型的称为“热狗折叠”的保守结构域，并且N-区域不完整，而C-区域是完整的“热狗折叠”；MaoC通过四个α螺旋相互作用形成了同源二聚体结构，且参与二聚化作用的氨基酸在R型水合酶中是高度保守的；结构上的精细分析与比对还发现MaoC存在一段潜在的活性抑制片段；
3.以序列和结构比对为基础，确定了MaoC的催化中心为以下三个氨基酸：D194、H199和G217。为了验证其催化作用我们做了三个定点突变：D194E、H199Q和G217A；对三个突变体重组蛋白进行了纯化，并对纯蛋白进行了圆二色性、蛋白杂交和分子排阻层析验证，以确定其分子量及蛋白的真实性等；酶活测定结果显示三个氨基酸中D194和H199对MaoC行使酶活性起重要作用，G217起次要作用，具体作用方式为：D194通过吸引催化水分子上的一个质子而激活水分子，活化了的水分子攻击底物巴豆酰辅酶A上的碳原子，同时H199对底物上的碳原子贡献一个质子以使反应顺利进行，而G217通过氢键作用与底物硫酯键上的碳酰基结合，起到稳定底物的作用；
4.二聚体是MaoC的活性形式，MaoC通过四个α螺旋相互作用形成了同源二聚体结构。通过序列和结构比对分析发现，参与二聚化作用的氨基酸在不同的R型水合酶中是高度保守的，同时大部分已报道的R型水合酶结构均为二聚体结构，并且与MaoC具有类似的作用方式。基于结构上的分析与比对，我们推测二聚化作用对MaoC行使酶活性是必须的。为了验证这一推测，我们做了三个定点突变：R23D，M27E和L191D；对三个突变体纯蛋白进行分子排阻层析验证表明三个突变体均成功破坏了MaoC的二聚化作用，成为单体蛋白；酶活测定结果显示三个突变体单体蛋白均丧失活性，证实二聚化作用对MaoC行使酶活性是必须的；本研究首次通过生化实验证实二聚化作用对MaoC酶活起关键作用，考虑到二聚化作用在R型水合酶类蛋白上的普遍存在性，我们的结论即二聚化作用对其行使正常酶活是必须的可能对此类蛋白具有普遍适用性；一个单体结构可能会更好的解释其机制，然而我们对MaoC单体蛋白的结晶实验未能成功；
5.以结构为基础我们对MaoC进行了酶工程改造，以期得到与野生型蛋白相比具有较高酶活的突变体。结构分析表明一段非保守的插入片段可能对MaoC的酶活调节起作用。如果我们的假设正确的话，删除此片段将会对MaoC的活性产生影响。基于此假设我们对此区域进行了定点突变与片段删除突变，并对纯化后的突变体重组蛋白进行了酶活测定，结果显示两个突变体分别有较大幅度的活性提高：突变体Del63-71酶活为91.2U/mg，约为野生型蛋白的1.5倍；突变体Del63-88酶活为126.7U/mg，约为野生型蛋白的两倍；而定点突变对MaoC酶活基本无影响；分子排阻层析实验表明突变体活性的增强与其聚合状态无关，因为两个活性增强突变体均为二聚体；我们的研究表明MaoC上非保守的插入片段对其活性有抑制作用。
随着世界环境问题变得越来越严峻，发展绿色环保塑料以改善环境迫在眉睫。在本研究中，我们报道了一个与PHA生物合成相关的新型水合酶结构，并且对其活性调节机制进行了研究与改进，这些研究将为以更低的成本生产PHA提供有效信息。
Phytophthora capsici is a worldwide soil borne disease. Pepper Phytophthora blight hasserious impact on pepper production all over the world, and it causes huge economic losses. Ithas a broad host range including pepper, pumpkin, cucumber, eggplant, tomato, and so on, upto more than20kinds of plants. In recent years there are many reports on its pathogenesis,pathogenic mechanism and control strategy, while there are few reports on searching for somebeneficial genes to serve mankind from such pathogens.
In2008, the whole-genome draft sequence data from Photophthora capsici was madeavailable and updated constantly on line(http://genome.jgi-psf.org/Phyca11/Phyca11.home.html) by U.S. Department of Energy JointGenome Institute-DOE JGI. By bioinformatic analysis, more and more interesting genes arebeing found and this benefits us to reveal its function and be used by mankind. In our study,MaoC-like hydratase (to be shorten for MaoC), is one of such genes based on analysis of thegenome sequences. MaoC-like hydratase (MaoC), which belongs to (R)-hydratase involved inlinking the β-oxidation and the polyhydroxyalkanoates (PHAs) biosynthetic pathways, hasbeen identified recently.
Problems concerning with the global environment have arisen much interest in thedevelopment of biodegradable polymers. Among the several biodegradable polymers underresearch, PHAs are good candidates for “green plastic” due to their biodegradable,biocompatible, thermoplastic, and mechanical properties. Because of this, considerableresearch has been undertaken to develop low cost and high efficiency processes for theproduction of PHAs. Reducing the cost of PHAs production may make PHA competitive as a“green plastic” alternative to conventional plastics which are difficult to degrade underrandom conditions. MaoC, possessing activity of (R)-hydratase involved in linking theβ-oxidation and the PHA biosynthetic pathways, has been recently identified in the fadBmutant Escherichia coli strain. MaoC is a new (R)-hydratase that catalyzes the (R)-specifichydration of the β-oxidation intermediate2-trans-enoyl-CoA to (R)-3-hydroxyacyl-CoA. ThusMaoC is an important enzyme in the biosynthesis of PHA because it supplies monomers thatare subsequently polymerized to form PHA by PHA synthase.
We cloned a new MaoC gene from Photophthora capsici, and the crystal structure of theenzyme has been resolved at high resolution. The detailed function has been clarified on thebasis of the structure, showing as follows:
1. We cloned a new beneficial gene named MaoC from pathogen Phytophthora capsici. MaoC was over-expressed in Escherichia coli and the recombinant MaoC was purified usingaffinity chromatography, anion-exchange chromatography and gel filtration chromatography.The purified recombinant MaoC was used to determine the Enoyl-CoA hydratase activityusing Crotonyl-CoA as the substrate, and the purified tagged MaoC showed the Enoyl-CoAhydratase activity of58.1U/mg towards Crotonyl-CoA. MaoC is a new member of(R)-hydratases which have been involved in biosynthesis of PHA, functioning as catalyzingthe (R)-specific hydration of the β-oxidation intermediate2-trans-enoyl-CoA to(R)-3-hydroxyacyl-CoA;
2. MaoC was crystallized using the hanging drop vapour diffusion method, and thecrystal structure of the enzyme was resolved at1.93resolution by molecular replacement.The detailed description and analysis of three-dimensional structure were carried out: Thestructure shows that MaoC has a canonical (R)-hydratase folding with an N-domain and aC-domain, located between them is the intervening bridge region. The crystal structure of theenzyme belongs to the so-called “hot dog” fold superfamily, compared to the C-domain, theN-domain has an incomplete hot dog fold. MaoC forms a homodimer that is mediated by fourshort α-helices from each monomeric MaoC, and the residues participating in dimerization arehighly conserved among the (R)-hydratases. Further analysis of the structure reveals that thereis a hypothetical inhibitory segment in regulating the activity of MaoC.
3. The active sites of MaoC (Asp-194, His-199and Gly-217) were identified by sequenceand structure alignment. To examine the importance of Asp-194, His-199and Gly-217forcatalysis, three mutants, D194N, H199Q, and G217A, were prepared and purified. To confirmthe presence and the apparent molecular mass of the purified mutant enzymes, circulardichroism, western blotting and gel filtration chromatography were carried out. The results ofactivity assay of the purified mutants show that Asp-194and His-199play a crucial role incatalysis, while Gly-217may be less important for catalysis. We propose that the threeresidues mentioned above play respective roles in the catalytic reaction of the enzyme asfollows. Asp-194may activate a water molecule by abstracting a proton from a watermolecule. The activated water molecule then attacks the carbon atom of crotonyl-CoA. Inaddition, His-199perhaps cooperatively donates a proton to the carbon atom of the substrate.Gly-217may hydrogen-bond to the carbonyl group of the thioester bond of the substrate.
4. In its structure, MaoC forms a homodimer that is mediated by four short α-helices fromeach monomeric MaoC. Based on the sequence and structure alignment, the residuesparticipating in dimerization are highly conserved among the (R)-hydratases, and most of thecrystal structures of hydratases resolved thus far are dimers with a similar interface to that in MaoC. These structural observations suggest that the dimeric MaoC is important for itsfunction. Supporting this conclusion, three mutations (R23D, M27E and L191D) disruptingthe dimerization of MaoC resulted in monomeric MaoC proteins in solution as indicated by agel filtration assay. More importantly, the enzymatic activities were completely absent in theMaoC mutant proteins. Together, these data strongly support the notion that MaoC must bedimerized to function. Our study provides the first biochemical evidence for the functionalsignificance of the dimer. A crystal structure of the monomeric MaoC will provide significantinsight into the mechanism underlying the requirement of MaoC dimerization for itsenzymatic activity. Unfortunately, all the efforts to crystallize a monomeric MaoC proteinfailed.
5. Based on the crystal structure, we made several mutants in engineering MaoC enzymeactivity in order to obtain mutants with higher activity compared to native MaoC. Structuralobservation suggests that a non-conserved insertion may have a role in regulating theenzymatic activity of MaoC. If this is the case, deletion of the insertion would generate aneffect on MaoC’s activity. To examine this possibility, we made single and deletion mutationssurrounding the hypothetical inhibitory segment and then assayed their effect on MaoC’sactivity. In full support of our prediction, one of the deletion mutant proteins, Del63-71,exhibited an activity of91.2U/mg in hydrolyzing enoyl-CoA, about1.5times that of the wildtype protein. Further deletion of the amino acids around the insertion (Del63-88) produced amore striking effect on promoting MaoC’s activity (126.7U/mg). In contrast, single mutationsin the insertion generated no significant effect on the activity of MaoC. The increased activityof the deletion mutant proteins was not caused by perturbation of the oligomerization status ofMaoC, because they both were eluted at a similar position to the wild type protein insize-exclusion chromatography. Collectively, our results indicate that an insertion region ofMaoC plays a role in inhibiting its activity.
As the environmental problem is becoming more and more serious all over the world, it isobviously significant to develop the “green plastic” to improve the environment. Here wereport the crystal structure of a new enzyme involved in PHA biosynthesis. The data in ourstudy reveal the regulatory mechanism of an (R)-hydratase, providing information on enzymeengineering to produce low cost PHAs.
引文
丁建成，戚仁德，顾江涛，等.（2001）.辣椒疫病发生原因与防治对策.安徽农业科学29（5），651-652.
付丽.（2012）.辣椒疫霉（Phytophthora capsici）果胶裂解酶基因克隆及功能研究.山东农业大学硕士学位论文.
高军.（2010）.辣椒疫病的发生及防治.西北园艺.
高智谋，郑小波，陆家云.（1999）.疫霉菌对杀菌剂抗性遗传研究（综述）.安徽农业大学学报26（2），155-161.
黄海东，卢显芝，熊诵锦，郝丽萍.（2006）.活性污泥中PHAMCL合成菌的筛选及特性研究.农业环境科学学报25（2），517-521.
李爱萍，李光吉.（2004）.聚羟基脂肪酸酯生物合成的研究进展.高分子通报5，20-27.
李兰芬，南洁，苏晓东.（2009）.蛋白质晶体学技术的发展与展望.生物物理学报23（4），246-255.
李美荣，刘永峰，聂亚峰，等.（2009）.辣椒疫病拮抗菌的分离、筛选与评价.长江蔬菜10，60-64.
罗赫荣，谢丙炎，马凤海（1999）.辣椒疫霉对甲霜灵和霜脲氰抗药性遗传的研究.湖南农业大学学报25（1），52-56.
刘小青，朱锦.（2011）.有关“环境友好塑料”几个概念的澄清.高分子通报6，100-102.
林亚静，刘志杰，龚为民.（2007）.蛋白质结构研究.生命科学19（3）,289-293.
凌志洋，刘望夷.（2009）.核糖体的研究历程-2009年诺贝尔化学奖简介.自然杂志31
（6），337-341.
马辉刚.（1988）.云南辣椒疫病菌种的鉴定.云南农业大学学报3(2)，125-132.
梅新兰，赵青云，谭石勇，等.（2010）.辣椒疫病拮抗菌株筛选、鉴定及其防效.应用生态学报21（10），2652-2658.
欧雄常，柳凤，詹儒林，等.（2009）.拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定.微生物学通报36（2），175-180.
戚仁德，汪涛，高智谋，等.（2011）.抗甲霜灵辣椒疫霉菌株的生物学特性.植物保护学报38（5），449-454.
邵见阳，马辉刚，苏玲.（1988）.辣椒疫霉病菌对内吸性杀菌剂抗药性的研究.江西农业学报10（4），46-50.
王会征.（2010）.辣椒疫霉两个重要基因（Pcpel2和MaoC-like dehydratase）的功能及晶体学研究.山东农业大学硕士学位论文.
王文广.（2011）.生物塑料和降解塑料的研究进展.塑料科技39（5），95-98.
王晓敏，巩振辉，逯红栋，李大伟.（2006）.辣椒疫霉菌孢子诱导技术研究.西北农业学报15（2），59-62.
王志田，史载诚，赵林安，等.（1993）.新疆辣椒疫病菌鉴定及生物学特性研究.新疆农业科学4，164-167.
郑军荣，周鹏，洪葵.（2003）. phaC1、phaC2基因双价植物表达载体的构建及其转基因烟草的获得.分子植物育种1（1），58-65.
郑世发.（2011）.辣椒栽培常易发生的问题及解决办法（一）.植保技术.
张景昱，叶梁，李丽，宋燕茹.（2001）.二次转化获得整合phba phbb phbc基因的转基因烟草.植物学报43(1)，59-26.
张向南.（2011）.聚3-羟基丁酸酯4-羟基丁酸酯性能研究.塑料科技39（5），57-62.
张运法.（2010）.X射线晶体学的诞生及其作用.化学工程与装备5，132-133.
(1994). The CCP4suite: programs for protein crystallography. Acta CrystallographicaSection D Biological Crystallography50(5),760-763.
Agnihotri, G., and Liu, H. W.(2003). Enoyl-CoA Hydratase Reaction, Mechanism, andInhibition. Bioorganic Medicinal Chemistry11,9-20.
Akey, D. L., Razelun, J. R., Tehranisa, J., Sherman, D. H., Gerwick, W. H., and Smith, J. L.(2010). Crystal Structures of Dehydratase Domains from the Curacin PolyketideBiosynthetic Pathway. Structure18(1),94-105.
Anderson, A. J., and Dawes, E. A.(1990). Occurrence, metabolism, metabolic role, andindustrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews54,450-472.
Arcy, A. D., Villard, F., and Marsh, M.(2007). An automated microseed matrix-screeningmethod for protein crystallization. Acta Crystallographica Section D BiologicalCrystallography63(4),550-554.
Argos, P., and Rossmann, M. G.(1980). Molecular replacement method. In: Ladd MFCRAP,eds, ed. Theory and practice of direct methods in crystallography New York: Plenumpublishing corp.361-417.
Benning, M. M., Wesenberg, G., Liu, R., Taylor, K. L., Dunaway-Mariano, D., and Holden, H.M.(1998). The three-dimensional structure of4-hydroxybenzoyl-CoA thioesterase fromPseudomonas sp. Strain CBS-3. Journal of Biological Chemistry273,33572-33579.
Bergfors, T.(2003). Seeds to crystals. Journal of Structural Biology142,66-76.
Bernd, H. A. R., and Alexander, S.(1999). Biochemical and genetic analysis of PHAsynthases and other proteins required for PHA synthesis. International Journal ofBiological Macromolecules25,3-19.
Bernd, H. A., Timothy, A., and Alexander, S.(2001). Role of fatty acid de novo biosynthesisin polyhydroxyalkanoic acid and rhamnolipid synthesis by Pseudomonads: establishmentof the transacylase (phaG)-nediated pathway for PHA biosynthesis in Escherichia coli.Applied and Environmental Microbiology67(7),3102-3109.
Bhambra, G. K., Wang, Z. Y., Soanes, D. M., Wakley, G.. E., and Talbot, N. J.(2006).Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphaeduring plant infection by Magnaporthe grisea. Molecular Microbiology1,46-60.
Bianchetti, C. M., Yi, L., Ragsdale, S. W., and Phillips, G. N.(2007). Comparison of Apo-andHeme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2. Journal ofBiological Chemistry282(52),37624-37631.
Bokhove, M., Yoshida, H., Hensgens, C. M. H., Metske van der Laan, J., Sutherland, J. D.,and Dijkstra, B. W.(2010). Structures of an Isopenicillin N Converting Ntn-HydrolaseReveal Different Catalytic Roles for the Active Site Residues of Precursor and MatureEnzyme. Structure18(3),301-308.
Bombach, P., Richnow, H. H., K stner, M., and Fischer, A.(2010). Current approaches forthe assessment of in situ biodegradation. Applied Microbiology and Biotechnology86,839-852.
Bradford, M. M.(1976). A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. AnalyticalBiochemistry72,248-254.
Buerger, M. J.(1950). Limitation of electron density by the patterson function. Proceedings ofthe National Academy of Sciences of USA36(12),738-742.
Byrom, D.(1992). Production of poly-β-hydroxybutyrate: Poly-β-hydroxyvaleratecopolymers. FEMS Microbiology Reviews103,247-250.
Cai, L., Yuan, M., Liu, F., Jian, J., and Chen, G.(2009). Enhanced production ofmedium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockoutmutant of Pseudomonas putida KT2442. Bioresource Technology100(7),2265-2270.
Campbell, S. J., Edwards, R. A., and Glover, J. N. M.(2010). Comparison of the Structuresand Peptide Binding Specificities of the BRCT Domains of MDC1and BRCA1.Structure18(2),167-176.
Castell, A., Johansson, P., Unge, T., Jones, T. A., and B ckbro, K.(2005). Rv0216, aconserved hypothetical protein from Mycobacterium tuberculosis that is essential forbacterial survival during infection, has a double hotdog fold. Protein Science14,1850-1862.
Chayen, N. E., Saridakis, E., and Sear, R. P.(2006). Experiment and theory for heterogeneousnucleation of protein crystals in a porous medium. Proceedings of the National Academyof Sciences of USA103(3),597-601.
Chen, G. Q., and Wu, Q.(2005). The application of polyhydroxyalkanoates as tissueengineering materials. Biomaterials26(33),6565-6578.
Chen, J., Zhang, L., Chen, J., and Chen, G.(2007). Biosynthesis and Characterization ofPolyhydroxyalkanoate Copolyesters in Ralstonia eutropha PHB4Harboring aLow-Substrate-Specificity PHA Synthase PhaC2Ps from Pseudomonas stutzeri1317.Chinese Journal of Chemical Engineering15(3),391-396.
Chen, Y., Dey, R., and Chen, L.(2010). Crystal Structure of the p53Core Domain Bound to aFull Consensus Site as a Self-Assembled Tetramer. Structure18(2),246-256.
Chung, M. G., and Rhee, Y. H.(2012). Overexpression of the (R)-Specific Enoyl-CoAHydratase Gene from Pseudomonas chlororaphis HS21in Pseudomonas Strains for theBiosynthesis of Polyhydroxyalkanoates of Altered Monomer Composition. Bioscience,Biotechnology, and Biochemistry76(3),613-616.
Daniel, K., Edouard, J., and Yaacov, O.(2005). Ecological and agricultural significance ofbacterial polyhydroxyalkanoates. Critical Reviews in Microbiology31,55-67.
Delano, W. L., and Brunger, A. T.(1995). The direct rotation function: Patterson correlationsearch applied to molecular replacement. Acta Crystallographica Section D BiologicalCrystallography51(5),740-748.
Diana, H., and Hsueh-Chia, C.(2008). Ac field enhanced protein crystallization. AppliedPhysics Letters92(22),223902.
Doi, Y., Kitamura, S., and Abe, H.(1995). Microbial synthesis and characterization ofpoly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules28,4822-4828.
Drenth, J.(1999). Principles of protein X-ray crystallography (2nd edition). Springeradvanced texts in chemistry New York: Springer xv,341p.
Emsley, P., and Cowtan, K.(2004). Coot: model-building tools for molecular graphics. ActaCrystallographica Section D Biological Crystallography60,2126-2132.
Engel, C., and Wierenga, R.(1996). The diverse world of coenzyme A binding proteins.Current Opinion Structural Biology6,790-797.
Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K.(1996). Crystalstructure of enoyl-coenzyme A (CoA) hydratase at2.5resolution: a spiral fold definesthe CoA-binding pocket. The EMBO Journal15,5135-5145.
Erdemir, D., Lee, A. Y., and Myerson, A. S.(2009). Nucleation of crystals from solution:classical and two-step models. Accounts of Chemical Research42(5),621-629.
Fanning, A. S., Lye, M. F., Anderson, J. M., and Lavie, A.(2007). Domain Swapping withinPDZ2Is Responsible for Dimerization of ZO Proteins. Journal of Biological Chemistry282(52),37710-37716.
Fiedler, S., Steinbüchel, A., and Rehm, B.(2002). The role of the fatty acid β-oxidationmultienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoatebiosynthesis: molecular characterization of the fadBA operon from P. oleovorans and ofthe enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida.Archives of Microbiology178(2),149-160.
Fogg, M. J., and Wilkinson, A. J.(2008). Higher-throughput approaches to crystallization andcrystal structure determination. Biochemical Society Transactions36(4),771-775.
Fukui, T., and Doi, Y.(1997). Cloning and analysis of thepoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonascaviae. Journal of Bacteriology179,4821-4830.
Fukui, T., Shiomi, N., and Doi, Y.(1998). Expression and characterization of (R)-specificenoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis byAeromonas caviae. Journal of Bacteriology180,667-673.
Fukui, T., Yokomizo, S., Kobayashi, G., and Doi, Y.(1999). Co-expression ofpolyhydroxyalkanoate synthase and (R)-enoyl-CoA hydratase genes of Aeromonascaviae establishes copolyester biosynthesis pathway in Escherichia coli. FEMSMicrobiology Letters170,69-75.
Garcia-Ruiz, L. M.(2003). Nucleation of protein crystals. Journal of Structural Biology142(1),22-31.
Gevens A. J., Donahoo R. S. and Lamour K. H.(2008). Characterization of Phytophthoracapsici Causing Foliar and PodGlight of Snap Bean in Michigan. Plant Disease92(2),201-209.
Goult, B. T., Bouaouina, M., Elliott, P. R., Bate, N., Patel, B., Gingras, A. R., Grossmann, J.G., Roberts, G. C. K., Calderwood, D. A., Critchley, D. R., and Barsukov, I. L.(2010).Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation.The EMBO Journal29(6),1069-1080.
Grage, K., Jahns, A. C., Parlane, N., Palanisamy, R., Rasiah, I. A., Atwood, J. A., and Rehm,B. H. A.(2009). Bacterial polyhydroxyalkanoate granules: Biogenesis, structure, andpotential use as nano-/micro-beads in biotechnological and biomedical applications.Biomacromolecules10(4),660-669.
Hahn, J. J., Eschenlauer, A. C., Sleytr, U. B., Somers, D. A., and Srienc, F.(1999).Peroxisomes as sites for synthesis of polyhydroxyalkanoates in transgenic plants.Biotechnology Progress15(6),1053-1057.
Hamada, H., Arakawa, T., and Shiraki, K.(2009). Effect of additives on protein aggregation.Current Pharmaceutical Biotechnology10(4),400-407.
Han, M.-J., Lee, J. W., Lee, S. Y., and Yoo, J. S.(2008). Proteome-Level Responses ofEscherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter inProtein Production. Journal of Biomedicine and Biotechnology2008,1-12.
Hansen, M., Hsu, A.-L., Dillin, A., and Kenyon, C.(2005). New Genes Tied to Endocrine,Metabolic, and Dietary Regulation of Lifespan from a Caenorhabditis elegans GenomicRNAi Screen. PLoS Genetics1(1), e17.
Hennig, S., Strauss, H. M., Vanselow, K., Yildiz,., Schulze, S., and Arens, J.(2009).Structural and functional analyses of PAS domain interactions of the clock proteinsDrosophila PERIOD and mouse PERIOD2. Plos Biology7, e1000094.
Hiroyasu, S., Takashi, M., and Tomonori, M.(1999). PHA production by activated sludge.International Journal of Biological Macromolecules25,105-109.
Hisano, T., Tsuge, T., Fukui T., Iwata, T., Miki, K., and Doi, Y.(2003). Crystal structure of the(R)-specific enoyl-CoA hydratase from Aeromonas caviae involved inpolyhydroxyalkanotes biosynthesis. Journal of Biological Chemistry278,617-624.
Hoffmann, N., Amara, A. A., Beermann, B. B., Qi, Q., Hinz, H. J., and Rehm, B. H.(2002).Biochemical characterization of the pseudomonas putida3-hydroxyacyl ACP: CoAtransacylase, which diverts intermediates of fatty acid de novo biosynthesis. Journal ofBiological Chemistry277(45),42926-42936.
Hoffmann, N., Steinbüchel, A., and Rehm, B. H. A.(2000). The Pseudomonas aeruginosaphaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consistingof medium-chain-length constituents from non-related carbon sources. FEMSMicrobiology Letters184,253-259.
Holm, L., and Sander, C.(1994). Searching protein structure databases has come of age.Proteins19,165-173.
Houmiel, K. L., Slater, S., Broyles, D., Casagrande, L., Colburn, S., Gonzalez, K., Mitsky, T.A., Reiser, S. E., Shah, R. D., Taylor, N. B., Tran, M., Valentin, H. E., and Gruys, K. J.(1999). Poly-(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus.Planta209,547-550.
Huang, Z., Feng, Y., Chen, D., Wu, X., Huang, S., Wang, X., Xiao, X., Li, W., Huang, N., Gu,L., Zhong, G., and Chai, J.(2008). Structural basis for activation and inhibition of thesecreted Chlamydia protease CPAF. Cell Host Microbe4,529-542.
Hubbard, P. A., Yu, W., Schulz, H., and Kim, J.-J. P.(2009). Domain swapping in thelow-similarity isomerase/hydratase superfamily: The crystal structure of ratmitochondrial Δ3, Δ2-enoyl-CoA isomerase. Protein Science14(6),1545-1555.
Hurtado-Gonzaleso., Aragon-Caballero L. and Apaza-Tapia W.(2008). Survival and Spread ofPhytophthora capsici in Coastal Peru. Phytopathology98(6),688-694.
Ito, L., Kobayashi, T., Shiraki, K. and Yamaguchi, H.(2008). Effect of amino acids and aminoacid derivatives on crystallization of hemoglobin and ribonuclease A. Journal ofSynchrotron Radiation15(3),316-318.
Jackson, K. L., Yin, J., Csinos, A. S., and Ji, P.(2010). Fungicidal activity of fluopicolide forsuppression of Phytophthora capsici on squash. Crop Protection29(12),1421-1427.
Jae Park, S., and Yup Lee, S.(2004). New fadB homologous enzymes and their use inenhanced biosynthesis of medium-chain-length polyhydroxyalkanoates infadBmutantEscherichia coli. Biotechnology and Bioengineering86(6),681-686.
Jancarik, J., and Kim, S. H.(1991). Sparse-matrix sampling-a screening method forcrystallization of proteins. Journal of applied crystallography24,409-411.
Jandrossek, D., Schirmer, A., and Schlegel, H. G.(1996). Biodegradation ofpolyhydroxyalkanoic acids. Applied Microbiology and Biotechnology46,451-463.
Jiang, Y., Hebly, M., Kleerebezem, R., Muyzer, G., and van Loosdrecht, M. C. M.(2011).Metabolic modeling of mixed substrate uptake for polyhydroxyalkanoate (PHA)production. Water Research45(3),1309-1321.
Jimena, A. R., Nancy, I. L., Ruben, O. F., and Beatriz, S. M.(2001). Polyhydroxyalkanoatedegradation is associated with nucleotide accumulation and enhances stress resistanceand survival of Pseudomonas oleovorans in natural water microcosms. Applied andEnvironmental Microbiology67(1),225-230.
John, M. E., and Keller, G.(1996). Metabolic pathway engineering in cotton: Biosynthesis ofpolyhydroxybutyrate in fiber cells. Proceedings of the National Academy of Sciences ofUSA93,12768-12773.
Kartha, G., and Parthasarathy, R.(1965). Combination of multiple isomorphous replacementand anomalous dispersion data for protein structure determination. Ⅰ. Determination ofheavy-atom positions in protein derivatives. Acta Crystallographica18(4),745-749.
Kartha, G., and Parthasarathy, R.(1965). Combination of multiple isomorphous replacementand anomalous dispersion data for protein structure determination. Ⅱ. Correlation of theheavy-atom positions in different isomorphous protein crystals. Acta Crystallographica18(4),749-753.
Kartha, G.(1965). Combination of multiple isomorphous replacement and anomalousdispersion data for protein structure determination.3. Refinement of heavy atompositions by the least-squares method. Acta Crystallographica19(6),883-885.
Kawashima, Y., Cheng, W., Mifune, J., Orita, I., Nakamura, S., and Fukui, T.(2011).Characterization and Functional Analyses of R-Specific Enoyl Coenzyme A Hydratasesin Polyhydroxyalkanoate-Producing Ralstonia eutropha. Applied and EnvironmentalMicrobiology78(2),493-502.
Khanna, S., and Srivastava, A. K.(2005). Recent advances in microbialpolyhydroxyalkanoates. Process Biochemistry40(2),607-619.
Koski, M. K.(2004). A Two-domain Structure of One Subunit Explains Unique Features ofEukaryotic Hydratase2. Journal of Biological Chemistry279(23),24666-24672.
Kristian Koski, M., Haapalainen, A. M., Hiltunen, J. K., and Glumoff, T.(2005). CrystalStructure of2-Enoyl-CoA Hydratase2from Human Peroxisomal MultifunctionalEnzyme Type2. Journal of Molecular Biology345(5),1157-1169.
Langenbach, S., Rehm, B. H. A., and Steinbüchel, A.(1997). Functional expression of thePHA synthase gene phaC1from Pseudomonas aeruginosa in Escherichia coli results inpoly(3-hydroxyalkanoate) synthesis. FEMS Microbiology Letters150,303-309.
Laskowski, R. A., Moss, D. S., and Thornton, J. M.(1993). Main-chain bond lengths andbond angles in protein structures. Journal of Molecular Biology231,1049-1067.
Leaversuch, R.(1987). Industry weighs the need to make polymer degradable. ModernPlastics64,52-55.
Leduc, D., Battesti, A., and Bouveret, E.(2007). The Hotdog Thioesterase EntH (YbdB) Playsa Role In Vivo in Optimal Enterobactin Biosynthesis by Interacting with the ArCPDomain of EntB. Journal of Bacteriology189(19),7112-7126.
Leesong, M., Henderson, B. S., Gillig, J. R., Schwab, J. M., and Smith, J. L.(1996). Structureof a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturatedfatty acids: two catalytic activities in one active site. Structure4,253-264.
Lee, S. Y., and Chang, H. N.(1995). Production of poly-(hydroxyalkanoic acid). Advances inBiochemical Engineering/Biotechnology52,27-58.
Lee, S. Y., and Choi, J.(1999). Production and degradation of polyhydroxyalkanoates inwaste environment. Waste Management19,133-139.
Lee, S. Y.(1996). Bacterial polyhydroxyalkanoates. Biotechnology and Bioengineering49,1-14.
Lee, S. Y., Park, S. H., Lee, Y., and Lee, S. H.(2001). Production of chiral and other valuablecompounds from microbial polyesters. In: Doi, Y., Steinbüchel, A.(eds.), BiopolymersWiley-VCH, Weinheim, Germany375-88.
Leslie, A. G. W.(2006). The integration of macromolecular diffraction data. ActaCrystallographica Section D Biological Crystallography62,48-57.
Li, J., Derewenda, U., Dauter, Z., Smith, S., and Derewenda, Z. S.(2000). Crystal structure ofthe Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme.Nature Structural&Molecular Biology7,555-559.
Li, R., Zhang, H., and Qi, Q.(2007). The production of polyhydroxyalkanoates inrecombinant Escherichia coli. Bioresource Technology98(12),2313-2320.
Li, Y., Zhang, Z., Robinson, G. E., and Palli, S. R.(2007). Identification and Characterizationof a Juvenile Hormone Response Element and Its Binding Proteins. Journal ofBiological Chemistry282(52),37605-37617.
Lu, Q. H., Han, J., Zhou, L. G., Zhou, J., and Xiang, H.(2008). Genetic and biochemicalcharacterization of the Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase inHaloferax mediterranei. Journal of Bacteriology190,4173-4180.
Madison, L. L., and Huisman, G. W.(1999). Metabolic engineering ofpoly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and Molecular BiologyReviews63(1),21-53.
Main, P., and Rossmann, M. G.(1966). Relationship among structure factors due to identicalmolecules in different crystallographic environments. Acta Crystallographica21(1),67-72.
Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sanchez, R., Melo, F., and Sali, A.(2000).Comparative protein structure modeling of genes and genomes. Annual Review ofBiophysics and Biomolecular Structure29,291-325.
Mathieu, M., Zeelen, J. P., Pauptit, R. A., Erdmann, R., Kunau, W. H., and Wierenga, R. K.(1994). The2.8crystal structure of peroxisomal3-ketoacyl-CoA thiolase ofSaccharomyces cerevisiae: a five-layered αβαβα structure constructed from two coredomains of identical topology. Structure2(9),797-808.
Matthews, B. W.(1968). Solvent content of protein crystals. Journal of Molecular Biology33,491-497.
Morris, R. J., Perrakis, A., and Lamzin, V. S.(2003). ARP/wARP and automatic interpretationof protein electron density maps. Methods in Enzymology374,229-244.
Mothes, G., Babel, W.(1995). Methylobacterium rhodesianum MB126possesses twostereospecific crotonyl-CoA hydratase. Canadian Journal of Microbiology41,68-72.
Motin, V. L., Georgescu, A. M., Elliott, J. M., Hu, P., Worsham, P. L., Ott, L. L., Slezak, T. R.,Sokhansanj, B. A., Regala, W. M., Brubaker, R. R., and Garcia, E.(2002). GeneticVariability of Yersinia pestis Isolates as Predicted by PCR-Based IS100Genotyping andAnalysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD).Journal of Bacteriology184(4),1019-1027.
Murshudov, G. N., Vagin, A. A., and Dodson, E. J.(1997). Refinement of macromolecularstructures by the maximum-likelihood method. Acta Crystallographica Section DBiological Crystallography53(3),240-255.
Nanev, C. N.(2007). On the slow kinetics of protein crystallization. Crystal Growth&Design7(8),1533-1540.
Nawrath, C., Poirier, Y., and Somerville, C.(1994). Targeting of the polyhydroxybutyratebiosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels ofpolymer accumulation. Proceedings of the National Academy of Sciences of USA91,12760-12764.
Ozaki, S., and Katayama, T.(2009). DnaA structure, function, and dynamics in the initiationat the chromosomal origin. Plasmid62(2),71-82.
Park, S. J., Ahn, W. S., Green, P. R., and Lee, S. Y.(2001). Production ofpoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by metabolically engineeredEscherichia coli strains. Biomacromolecules2,248-254.
Park, S. J., and Lee, S. Y.(2003). Identification and Characterization of a New EnoylCoenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-LengthPolyhydroxyalkanoates in Recombinant Escherichia coli. Journal of Bacteriology185(18),5391-5397.
Park, S. J., Park, J. P., and Lee, S. Y.(2002). Metabolic engineering of Escherichia coli for theproduction of medium-chain-length polyhydroxyalkanoates rich in specific monomers.FEMS Microbiology Letters214,217-222.
Perrin, M. H., Grace, C. R. R., DiGruccio, M. R., Fischer, W. H., Maji, S. K., Cantle, J. P.,Smith, S., Manning, G., Vale, W. W., and Riek, R.(2007). Distinct Structural andFunctional Roles of Conserved Residues in the First Extracellular Domain of Receptorsfor Corticotropin-releasing Factor and Related G-protein-coupled Receptors. Journal ofBiological Chemistry282(52),37529-37536.
Phan, I., Subramanian, S., Olsen, C., Edwards, T. E., Guo, W., Zhang, Y., Van Voorhis, W. C.,Stewart, L. J., and Myler, P. J.(2011). Structure of fumarate hydratase fromRickettsiaprowazekii, the agent of typhus and suspected relative of the mitochondria. ActaCrystallographica Section F Structural Biology and Crystallization Communications67(9),1123-1128.
Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N., and Suguna, K.(2009). Analysis ofproteins with the “Hot dog” fold: Prediction of function and identification of catalyticresidues of hypothetical proteins. BMC Structural Biology9(1),1-16.
Poirier, Y., Dennis, D. E., Klomparens, K., and Somerville, C.(1992). Polyhydroxybutyrate, abiodegradable thermoplastic, produced in transgenic plants. Science256,520-523.
Putnam, N. H., Srivastava, M., Hellsten, U., Dirks, B., Chapman, J., Salamov, A., Terry, A.,Shapiro, H., Lindquist, E., Kapitonov, V. V., Jurka, J., Genikhovich, G., Grigoriev, I. V.,Lucas, S. M., Steele, R. E., Finnerty, J. R., Technau, U., Martindale, M. Q., and Rokhsar,D. S.(2007). Sea Anemone Genome Reveals Ancestral Eumetazoan Gene Repertoire andGenomic Organization. Science317(5834),86-94.
Qin, Y. M., Haapalainen, A. M., Kilpel inen, S. H., Marttila, M. S., Koski, M. K., Glumoff, T.,Novikov, D. K., and Hiltunen, J. K.(2000). Human peroxisomal multifunctional enzymetype2. Site-directed mutagenesis studies show the importance of two protic residues for2-enoyl-CoA hydratase2activity. Journal of Biological Chemistry275,4965-4972.
Qi, Q., Rehm, B. H. A., and Steinbüchel, A.(1997). Synthesis of poly(3-hydroxyalkanoates)in Escherichia coli expressing the PHA synthase gene phaC2from Pseudomonasaeruginosa: comparison of PhaC1and PhaC2. FEMS Microbiology Letters157,155-162.
Qi, Q., Steinbüchel, A., and Rehm, B. H. A.(1998). Metabolic routing towardspolyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition offatty acid beta-oxidation by acrylic acid. FEMS Microbiology Letters167,89-94.
Otwinowski, Z., and Minor, W.(1997). Processing of X-ray diffraction data collected inoscillation mode. Methods in Enzymology276,307-326.
Rayment, I.(1997). Reductive alkylation of lysine residues to alter crystallization propertiesof proteins. Methods in Enzymology276,171-179.
Read, R. J.(2001). Pushing the boundaries of molecular replacement with maximumlikelihood. Acta Crystallographica Section D Biological Crystallography57(10),1373-1382.
Rehm, B. H. A., Kriiger, N., and Steinbuchel, A.(1998). A new metabolic link between fattyacid de novo synthesis and polyhydroxyalkanoic acid synthesis. The Journal ofBiological Chemistry273,24044-24051.
Rehm, B. H. A.(2003). Polyester synthases: natural catalysts for plastics. BiochemicalJournal376,15-33.
Rehm, B. H.(2007). Biogenesis of microbial polyhydroxyalkanoate granules: a platformtechnology for the production of tailor-made bioparticles. Current Issues in MolecularBiology9(1),41-62.
Reiser, S. E., Mitsky, T. A., and Gruys, K. J.(2000). Characterization and cloning of an(R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use ofthis enzyme for PHA production in Escherichia coli. Applied Microbiology andBiotechnology53,209-218.
Ren, Q., Sierro, N., Witholt, B., and Kessler, B.(2000). FabG, an NADPHdependent3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors formedium-chain-length poly-3-hydroxyalkanoate biosynthesis in Escherichia coli. Journalof Bacteriology182,2978-2981.
Robert, L., Robert, A. M., and Jacotot, B.(1998). Elastin elastase atherosclerosis revisited.Atherosclerosis140,281-295.
Robles-Yerena, L., Rodríguez-Villarreal, R. A., Ortega-Amaro, M. A., Fraire-Velázquez, S.,Simpson, J., Rodríguez-Guerra, R., and Jiménez-Bremont, J. F.(2010). Characterizationof a new fungal antagonist of Phytophthora capsici. Scientia Horticulturae125(3),248-255.
Rossier, O. M., Gauthier, N., Biais, N., Vonnegut, W., Fardin, M.-A., Avigan, P., Heller, E. R.,Mathur, A., Ghassemi, S., Koeckert, M. S., Hone, J. C., and Sheetz, M. P.(2010). Forcegenerated by actomyosin contraction builds bridges between adhesive contacts. TheEMBO Journal29(6),1055-1068.
Rossmann, M. G., Blow, D. M., Harding, M. M., and Coller, E.(1964) The relative positionsof independent molecules within the same asymmetric unit. Acta Crystallographica17(4),338-342.
Sacco, E., Legendre, V., Laval, F., Zerbib, D., Montrozier, H., Eynard, N., Guilhot, C., Daffé,M., and Quémard, A.(2007). Rv3389C from Mycobacterium tuberculosis, a member ofthe (R)-specific hydratase/dehydratase family. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics1774(2),303-311.
Saridakis, E., and Chayen, N. E.(2009). Towards a “universal” nucleant for proteincrystallization. Trends in Biotechnology27(2),99-106.
Schena, L., and Cooke, D. E. L.(2006). Assessing the potential of regions of the nuclear andmitochondrial genome to develop a “molecular tool box” for the detection andcharacterization of Phytophthora species. Journal of Microbiological Methods67(1),70-85.
Scibetta, S., Schena, L., Chimento, A., Cacciola, S. O., and Cooke, D. E. L.(2012). Amolecular method to assess Phytophthora diversity in environmental samples. Journal ofMicrobiological Methods88(3),356-368.
Shandilya, S. M. D., Nalam, M. N. L., Nalivaika, E. A., Gross, P. J., Valesano, J. C., Shindo,K., Li, M., Munson, M., Royer, W. E., Harjes, E., Kono, T., Matsuo, H., Harris, R. S.,Somasundaran, M., and Schiffer, C. A.(2010). Crystal Structure of the APOBEC3GCatalytic Domain Reveals Potential Oligomerization Interfaces. Structure18(1),28-38.
Shi, R., Proteau, A., Villarroya, M., Moukadiri, I., Zhang, L. H., Trempe, J. F., Matte, A.,Armengod, M. E., and Cygler, M.(2010). Structural basis for Fe-S cluster assembly andtRNA thiolation mediated by IscS protein-protein interactions. Plos Biology8,e1000354.
Sholberg P. L., Walkerm C. O. and Gorman D. T.(2007). First Report of Phytophthora capsicion Cucurbits and Peppers in British Columbia. Canadian Journal of Plant Pathology29(2),153-158.
Shrivastav, A., Mishra, S. K., and Mishra, S.(2010). Polyhydroxyalkanoate (PHA) synthesisby Spirulina subsalsa from Gujarat coast of India. International Journal of BiologicalMacromolecules46(2),255-260.
Silke, F., Alexander, S., and Bernd, H. A.(2000). PhaG-mediated synthesis of poly-3-hydroxyalkanoates consisting of medium-chain–length constituents from nonrelatedcarbon sources in recombinant pseudomonas fragi. Applied and EnvironmentalMicrobiology66(5),2117-2124.
Studholme, D. J., and Dixon, R.(2003). Domain Architectures of54-DependentTranscriptional Activators. Journal of Bacteriology185(6),1757-1767.
Sundaramoorthy, R., Micossi, E., Alphey, M. S., Germain, V., Bryce, J. H., Smith, S. M.,Leonard, G. A., and Hunter, W. N.(2006). The crystal structure of a plant3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acidbeta-oxidation. Journal of Molecular Biology359(2),347-357.
Taguchi, K., Aoyagi, Y., Matsusaki, H., Fukui, T., and Doi, Y.(1999). Co-expression of3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHAproduction in Escherichia coli HB101strain. FEMS Microbiology Letters176,183-190.
Taguchi, S., Nakamura, H., Kichise, T., Tsuge, T., Yamato, I., and Doi, Y.(2003). Productionof polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinantRalstonia eutropha using mutants of original PHA synthase. Biochemical EngineeringJournal16(2),107-113.
Takayama, K., Wang, C., and Besra, G. S.(2005). Pathway to Synthesis and Processing ofMycolic Acids in Mycobacterium tuberculosis. Clinical Microbiology Reviews18(1),81-101.
Takehara, M., Ino, K., Takakusagi, Y., Oshikane, H., Nureki, O., Ebina, T., Mizukami, F., andSakaguchi, K.(2008). Use of layer silicate for protein crystallization: effects ofmicromica and chlorite powders in hanging drops. Analytical Biochemistry373(2),322-329.
Taylor, G.(2003). The phase problem. Acta Crystallographica Section D BiologicalCrystallography59(11),1881-1890.
Thakur, A. S., Robin, G., Guncar, G., Saunders, N. F. W., Newman, J., Martin, J. L., and Kobe,B.(2007). Improved success of sparse matrix protein crystallization screening withheterogeneous nucleating agents. Plos one2(10),1091.
Tian, X., Wang, A., Qiao, S., Zhang, G., Xi, J., Li, X., Liu, Y., and Xu, Y.(2009). Expression,purification and characterization of a functional extracellular domain of porcine FcγRII.Protein Expression and Purification68(1),12-17.
Tmaietti G. and Valentino D.(2001). Physiological Characterisation of a Population ofPhytophthora capsici Leon.from Northern Italy. Journal of Plant Pathology83(3),199-205.
Tollin, P., and Rossmann, M. G.(1966). A description of various rotation function programs.Acta Crystallographica21(6),872-876.
Tollin, P., Main, P., and Rossmann, M. G.(1966). The symmetry of the rotation function. ActaCrystallographica20(3),404-407.
Truong, N. V., Liew, E. C. Y., and Burgess, L. W.(2010). Characterisation of Phytophthoracapsici isolates from black pepper in Vietnam. Fungal Biology114(2-3),160-170.
Tsuchida, S., Kawamoto, K., Nunome, K., Hamaue, N., Hui, S. P., Murai, T., Aoki, Takashi.,and Kurosawa, T.(2011). Chiral Separation, Determination of Absolute Configuration,and High-performance Liquid Chromatography Detection of Enantiomeric3-hydroxyhexadecanoyl-CoA. Journal of Oleo Science60(2),87-92.
Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A., and Doi Y.(1999).Molecular cloning of two (R)-specific enoyl-CoA Hydratase genes from Pseudomonasaeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS MicrobiologyLetters184,193-198.
Tsuge, T., Hisano, T., Taguchi, S., and Doi, Y.(2003). Alteration of Chain Length SubstrateSpecificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratasethrough Site-Directed Mutagenesis. Applied and Environmental Microbiology69(8),4830-4836.
Tsuge, T., Taguchi, K., Taguchi, S., and Doi, Y.(2003). Molecular characterization andproperties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa:metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid β-oxidation.International Journal of Biological Macromolecules31,195-205.
Tyler, B. M., Tripathy, S., Zhang, X., Dehal, P., Jiang, R. H., Aerts, A., Arredondo, F. D.,Baxter, L., Bensasson, D., Beynon, J. L., Chapman, J., Damasceno, C. M., Dorrance, A.E., Dou, D., Dickerman, A. W., Dubchak, I. L., Garbelotto, M., Gijzen, M., Gordon, S.G., Govers, F., Grunwald, N. J., Huang, W., Ivors, K. L., Jones, R. W., Kamoun, S.,Krampis, K., Lamour, K. H., Lee, M. K., McDonald, W. H., Medina, M., Meijer, H. J.,Nordberg, E. K., Maclean, D. J., Ospina-Giraldo, M. D., Morris, P. F., Phuntumart, V.,Putnam, N. H., Rash, S., Rose, J. K., Sakihama, Y., Salamov, A. A., Savidor, A.,Scheuring, C. F., Smith, B. M., Sobral, B. W., Terry, A., Torto-Alalibo, T. A., Win, J., Xu,Z., Zhang, H., Grigoriev, I. V., Rokhsar, D. S., Boore, J. L.(2006). Phytophthoragenome sequences uncover evolutionary origins and mechanisms of pathogenesis.Science313(5791),1261-1266.
Uta, B., Yaroslav, T., Gotthard, K., and Wolfgang, B.(2002). Yeasts as producers ofpolyhydroxyalkanoates: Genetic engineering of Saccharomyces cerevisiae.Macromolecular Bioscience2(8),380-386.
Vagin, A., and Teplyakov, A.(1997). MOLREP: an automated program for molecularreplacement. Journal of Applied Crystallography30,1022-1025.
Valappil, S. P., Peiris, D., Langley, G. J., Herniman, J. M., Boccaccini, A. R., Bucke, C., andRoy, I.(2007). Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelatedcarbon sources by a newly characterized Bacillus spp. Journal of Biotechnology127(3),475-487.
Venkata Mohan, S., and Venkateswar Reddy, M.(2013). Optimization of critical factors toenhance polyhydroxyalkanoates (PHA) synthesis by mixed culture using Taguchi designof experimental methodology. Bioresource Technology128,409-416.
Vivian, J. P., Riedmaier, P., Ge, H., Le Nours, J., Sansom, F. M., Wilce, M. C. J., Byres, E.,Dias, M., Schmidberger, J. W., Cowan, P. J., d'Apice, A. J. F., Hartland, E. L., Rossjohn,J., and Beddoe, T.(2010). Crystal Structure of a Legionella pneumophila Ecto-Triphosphate Diphosphohydrolase, A Structural and Functional Homolog of theEukaryotic NTPDases. Structure18(2),228-238.
Voinova, O. N., Gladyshev, M. I., and Volova, T. G.(2008). Comparative study of PHAdegradation in natural reservoirs having various types of ecosystems. MacromolecularSymposia269,34-37.
Volova, T. G., Boyandin, A. N., Vasiliev, A. D., Karpov, V. A., Prudnikova, S. V., Mishukova,O. V., Boyarskikh, U. A., Filipenko, M. L., Rudnev, V. P., Bá Xuan, B., Vi t D ng, V.,and Gitelson, I. I.(2010). Biodegradation of polyhydroxyalkanoates (PHAs) in tropicalcoastal waters and identification of PHA-degrading bacteria. Polymer Degradation andStability95(12),2350-2359.
Volova, T. G., Gladyshev, M. I., Trusova, M. Y., and Zhila, N. O.(2006). Degradation ofpolyhydroxyalkanoates and the composition of microbial destructors under naturalconditions. Microbiology75(5),593-598.
Volova, T. G., Gladyshev, M. I., Trusova, M. Y., and Zhila, N. O.(2007). Degradation ofpolyhydroxyalkanoates in eutrophic reservoir. Polymer Degradation and Stability92,580-586.
Vo, M. T., Lee, K.-W., Jung, Y.-M., and Lee, Y.-H.(2008). Comparative effect ofoverexpressed phaJ and fabG genes supplementing (R)-3-hydroxyalkanoate monomerunits on biosynthesis of mcl-polyhydroxyalkanoate in Pseudomonas putida KCTC1639.Journal of Bioscience and Bioengineering106(1),95-98.
Walter, T. S., Meier, C., Assenberg, R., Au, K. F., Ren, J., Verma, A., Nettleship, J. E., Owens,R. J., Stuart, D. I., and Grimes, J. M.(2006). Lysine methylation as a routine rescuestrategy for protein crystallization. Structure14(11),1617-1622.
Wang, Q., Xia, Y., Chen, Q., and Qi, Q.(2012). Incremental truncation of PHA synthasesresults in altered product specificity. Enzyme and Microbial Technology50(6-7),293-297.
Winn, M. D., Murshudov, G. N., and Papiz, M. Z.(2003). Macromolecular TLS refinement inREFMAC at moderate resolutions. Methods in Enzymology374,300-321.
Wu, L., Wang, L., Hua, G., Liu, K., Yang, X., Zhai, Y., Bartlam, M., Sun, F., and Fan, Z.(2009). Structural Basis for Proteolytic Specificity of the Human Apoptosis-InducingGranzyme M. The Journal of Immunology183(1),421-429.
Xu, H., Shi, Y., Wang, J., Jones, D., Weilrauch, D., Ying, R., Wakim, B., and Pritchard, K. A.(2007). A Heat Shock Protein90Binding Domain in Endothelial Nitric-oxide SynthaseInfluences Enzyme Function. Journal of Biological Chemistry282(52),37567-37574.
Xu, J., Zhao, X., Wang, X., Zhao, Z., and Du, Y.(2007). Oligochitosan inhibits Phytophthoracapsici by penetrating the cell membrane and putative binding to intracellular targets.Pesticide Biochemistry and Physiology88(2),167-175.
Yasui, N., Nogi, T., and Takagi, J.(2010). Structural Basis for Specific Recognition of Reelinby Its Receptors. Structure18(3),320-331.
You, Z., Omura, S., Ikeda, H., Cane, D. E., and Jogl, G.(2007). Crystal Structure of theNon-heme Iron Dioxygenase PtlH in Pentalenolactone Biosynthesis. Journal ofBiological Chemistry282(50),36552-36560.
Yücel S.(1995). A study on soil solarization and combined with fumigant application tocontrol Phytophthora crown blight (Phytophthora capsici Leonian) on peppers in theEast Mediterranean region of Turkey. Crop Protection14(8),653-655.
Zarzycki, J., Schlichting, A., Strychalsky, N., Muller, M., Alber, B. E., and Fuchs, G.(2007).Mesaconyl-Coenzyme A Hydratase, a New Enzyme of Two Central Carbon Metabolic
Pathways in Bacteria. Journal of Bacteriology190(4),1366-1374.
付丽.（2012）.辣椒疫霉（Phytophthora capsici）果胶裂解酶基因克隆及功能研究.山东农业大学硕士学位论文.
高军.（2010）.辣椒疫病的发生及防治.西北园艺.
高智谋，郑小波，陆家云.（1999）.疫霉菌对杀菌剂抗性遗传研究（综述）.安徽农业大学学报26（2），155-161.
黄海东，卢显芝，熊诵锦，郝丽萍.（2006）.活性污泥中PHAMCL合成菌的筛选及特性研究.农业环境科学学报25（2），517-521.
李爱萍，李光吉.（2004）.聚羟基脂肪酸酯生物合成的研究进展.高分子通报5，20-27.
李兰芬，南洁，苏晓东.（2009）.蛋白质晶体学技术的发展与展望.生物物理学报23（4），246-255.
李美荣，刘永峰，聂亚峰，等.（2009）.辣椒疫病拮抗菌的分离、筛选与评价.长江蔬菜10，60-64.
罗赫荣，谢丙炎，马凤海（1999）.辣椒疫霉对甲霜灵和霜脲氰抗药性遗传的研究.湖南农业大学学报25（1），52-56.
刘小青，朱锦.（2011）.有关“环境友好塑料”几个概念的澄清.高分子通报6，100-102.
林亚静，刘志杰，龚为民.（2007）.蛋白质结构研究.生命科学19（3）,289-293.
凌志洋，刘望夷.（2009）.核糖体的研究历程-2009年诺贝尔化学奖简介.自然杂志31
（6），337-341.
马辉刚.（1988）.云南辣椒疫病菌种的鉴定.云南农业大学学报3(2)，125-132.
梅新兰，赵青云，谭石勇，等.（2010）.辣椒疫病拮抗菌株筛选、鉴定及其防效.应用生态学报21（10），2652-2658.
欧雄常，柳凤，詹儒林，等.（2009）.拮抗辣椒疫病菌的红树内生细菌筛选及RS261菌株鉴定.微生物学通报36（2），175-180.
戚仁德，汪涛，高智谋，等.（2011）.抗甲霜灵辣椒疫霉菌株的生物学特性.植物保护学报38（5），449-454.
邵见阳，马辉刚，苏玲.（1988）.辣椒疫霉病菌对内吸性杀菌剂抗药性的研究.江西农业学报10（4），46-50.
王会征.（2010）.辣椒疫霉两个重要基因（Pcpel2和MaoC-like dehydratase）的功能及晶体学研究.山东农业大学硕士学位论文.
王文广.（2011）.生物塑料和降解塑料的研究进展.塑料科技39（5），95-98.
王晓敏，巩振辉，逯红栋，李大伟.（2006）.辣椒疫霉菌孢子诱导技术研究.西北农业学报15（2），59-62.
王志田，史载诚，赵林安，等.（1993）.新疆辣椒疫病菌鉴定及生物学特性研究.新疆农业科学4，164-167.
郑军荣，周鹏，洪葵.（2003）. phaC1、phaC2基因双价植物表达载体的构建及其转基因烟草的获得.分子植物育种1（1），58-65.
郑世发.（2011）.辣椒栽培常易发生的问题及解决办法（一）.植保技术.
张景昱，叶梁，李丽，宋燕茹.（2001）.二次转化获得整合phba phbb phbc基因的转基因烟草.植物学报43(1)，59-26.
张向南.（2011）.聚3-羟基丁酸酯4-羟基丁酸酯性能研究.塑料科技39（5），57-62.
张运法.（2010）.X射线晶体学的诞生及其作用.化学工程与装备5，132-133.
(1994). The CCP4suite: programs for protein crystallography. Acta CrystallographicaSection D Biological Crystallography50(5),760-763.
Agnihotri, G., and Liu, H. W.(2003). Enoyl-CoA Hydratase Reaction, Mechanism, andInhibition. Bioorganic Medicinal Chemistry11,9-20.
Akey, D. L., Razelun, J. R., Tehranisa, J., Sherman, D. H., Gerwick, W. H., and Smith, J. L.(2010). Crystal Structures of Dehydratase Domains from the Curacin PolyketideBiosynthetic Pathway. Structure18(1),94-105.
Anderson, A. J., and Dawes, E. A.(1990). Occurrence, metabolism, metabolic role, andindustrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews54,450-472.
Arcy, A. D., Villard, F., and Marsh, M.(2007). An automated microseed matrix-screeningmethod for protein crystallization. Acta Crystallographica Section D BiologicalCrystallography63(4),550-554.
Argos, P., and Rossmann, M. G.(1980). Molecular replacement method. In: Ladd MFCRAP,eds, ed. Theory and practice of direct methods in crystallography New York: Plenumpublishing corp.361-417.
Benning, M. M., Wesenberg, G., Liu, R., Taylor, K. L., Dunaway-Mariano, D., and Holden, H.M.(1998). The three-dimensional structure of4-hydroxybenzoyl-CoA thioesterase fromPseudomonas sp. Strain CBS-3. Journal of Biological Chemistry273,33572-33579.
Bergfors, T.(2003). Seeds to crystals. Journal of Structural Biology142,66-76.
Bernd, H. A. R., and Alexander, S.(1999). Biochemical and genetic analysis of PHAsynthases and other proteins required for PHA synthesis. International Journal ofBiological Macromolecules25,3-19.
Bernd, H. A., Timothy, A., and Alexander, S.(2001). Role of fatty acid de novo biosynthesisin polyhydroxyalkanoic acid and rhamnolipid synthesis by Pseudomonads: establishmentof the transacylase (phaG)-nediated pathway for PHA biosynthesis in Escherichia coli.Applied and Environmental Microbiology67(7),3102-3109.
Bhambra, G. K., Wang, Z. Y., Soanes, D. M., Wakley, G.. E., and Talbot, N. J.(2006).Peroxisomal carnitine acetyl transferase is required for elaboration of penetration hyphaeduring plant infection by Magnaporthe grisea. Molecular Microbiology1,46-60.
Bianchetti, C. M., Yi, L., Ragsdale, S. W., and Phillips, G. N.(2007). Comparison of Apo-andHeme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2. Journal ofBiological Chemistry282(52),37624-37631.
Bokhove, M., Yoshida, H., Hensgens, C. M. H., Metske van der Laan, J., Sutherland, J. D.,and Dijkstra, B. W.(2010). Structures of an Isopenicillin N Converting Ntn-HydrolaseReveal Different Catalytic Roles for the Active Site Residues of Precursor and MatureEnzyme. Structure18(3),301-308.
Bombach, P., Richnow, H. H., K stner, M., and Fischer, A.(2010). Current approaches forthe assessment of in situ biodegradation. Applied Microbiology and Biotechnology86,839-852.
Bradford, M. M.(1976). A rapid and sensitive method for the quantitation of microgramquantities of protein utilizing the principle of protein-dye binding. AnalyticalBiochemistry72,248-254.
Buerger, M. J.(1950). Limitation of electron density by the patterson function. Proceedings ofthe National Academy of Sciences of USA36(12),738-742.
Byrom, D.(1992). Production of poly-β-hydroxybutyrate: Poly-β-hydroxyvaleratecopolymers. FEMS Microbiology Reviews103,247-250.
Cai, L., Yuan, M., Liu, F., Jian, J., and Chen, G.(2009). Enhanced production ofmedium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockoutmutant of Pseudomonas putida KT2442. Bioresource Technology100(7),2265-2270.
Campbell, S. J., Edwards, R. A., and Glover, J. N. M.(2010). Comparison of the Structuresand Peptide Binding Specificities of the BRCT Domains of MDC1and BRCA1.Structure18(2),167-176.
Castell, A., Johansson, P., Unge, T., Jones, T. A., and B ckbro, K.(2005). Rv0216, aconserved hypothetical protein from Mycobacterium tuberculosis that is essential forbacterial survival during infection, has a double hotdog fold. Protein Science14,1850-1862.
Chayen, N. E., Saridakis, E., and Sear, R. P.(2006). Experiment and theory for heterogeneousnucleation of protein crystals in a porous medium. Proceedings of the National Academyof Sciences of USA103(3),597-601.
Chen, G. Q., and Wu, Q.(2005). The application of polyhydroxyalkanoates as tissueengineering materials. Biomaterials26(33),6565-6578.
Chen, J., Zhang, L., Chen, J., and Chen, G.(2007). Biosynthesis and Characterization ofPolyhydroxyalkanoate Copolyesters in Ralstonia eutropha PHB4Harboring aLow-Substrate-Specificity PHA Synthase PhaC2Ps from Pseudomonas stutzeri1317.Chinese Journal of Chemical Engineering15(3),391-396.
Chen, Y., Dey, R., and Chen, L.(2010). Crystal Structure of the p53Core Domain Bound to aFull Consensus Site as a Self-Assembled Tetramer. Structure18(2),246-256.
Chung, M. G., and Rhee, Y. H.(2012). Overexpression of the (R)-Specific Enoyl-CoAHydratase Gene from Pseudomonas chlororaphis HS21in Pseudomonas Strains for theBiosynthesis of Polyhydroxyalkanoates of Altered Monomer Composition. Bioscience,Biotechnology, and Biochemistry76(3),613-616.
Daniel, K., Edouard, J., and Yaacov, O.(2005). Ecological and agricultural significance ofbacterial polyhydroxyalkanoates. Critical Reviews in Microbiology31,55-67.
Delano, W. L., and Brunger, A. T.(1995). The direct rotation function: Patterson correlationsearch applied to molecular replacement. Acta Crystallographica Section D BiologicalCrystallography51(5),740-748.
Diana, H., and Hsueh-Chia, C.(2008). Ac field enhanced protein crystallization. AppliedPhysics Letters92(22),223902.
Doi, Y., Kitamura, S., and Abe, H.(1995). Microbial synthesis and characterization ofpoly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules28,4822-4828.
Drenth, J.(1999). Principles of protein X-ray crystallography (2nd edition). Springeradvanced texts in chemistry New York: Springer xv,341p.
Emsley, P., and Cowtan, K.(2004). Coot: model-building tools for molecular graphics. ActaCrystallographica Section D Biological Crystallography60,2126-2132.
Engel, C., and Wierenga, R.(1996). The diverse world of coenzyme A binding proteins.Current Opinion Structural Biology6,790-797.
Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K.(1996). Crystalstructure of enoyl-coenzyme A (CoA) hydratase at2.5resolution: a spiral fold definesthe CoA-binding pocket. The EMBO Journal15,5135-5145.
Erdemir, D., Lee, A. Y., and Myerson, A. S.(2009). Nucleation of crystals from solution:classical and two-step models. Accounts of Chemical Research42(5),621-629.
Fanning, A. S., Lye, M. F., Anderson, J. M., and Lavie, A.(2007). Domain Swapping withinPDZ2Is Responsible for Dimerization of ZO Proteins. Journal of Biological Chemistry282(52),37710-37716.
Fiedler, S., Steinbüchel, A., and Rehm, B.(2002). The role of the fatty acid β-oxidationmultienzyme complex from Pseudomonas oleovorans in polyhydroxyalkanoatebiosynthesis: molecular characterization of the fadBA operon from P. oleovorans and ofthe enoyl-CoA hydratase genes phaJ from P. oleovorans and Pseudomonas putida.Archives of Microbiology178(2),149-160.
Fogg, M. J., and Wilkinson, A. J.(2008). Higher-throughput approaches to crystallization andcrystal structure determination. Biochemical Society Transactions36(4),771-775.
Fukui, T., and Doi, Y.(1997). Cloning and analysis of thepoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonascaviae. Journal of Bacteriology179,4821-4830.
Fukui, T., Shiomi, N., and Doi, Y.(1998). Expression and characterization of (R)-specificenoyl coenzyme A hydratase involved in polyhydroxyalkanoate biosynthesis byAeromonas caviae. Journal of Bacteriology180,667-673.
Fukui, T., Yokomizo, S., Kobayashi, G., and Doi, Y.(1999). Co-expression ofpolyhydroxyalkanoate synthase and (R)-enoyl-CoA hydratase genes of Aeromonascaviae establishes copolyester biosynthesis pathway in Escherichia coli. FEMSMicrobiology Letters170,69-75.
Garcia-Ruiz, L. M.(2003). Nucleation of protein crystals. Journal of Structural Biology142(1),22-31.
Gevens A. J., Donahoo R. S. and Lamour K. H.(2008). Characterization of Phytophthoracapsici Causing Foliar and PodGlight of Snap Bean in Michigan. Plant Disease92(2),201-209.
Goult, B. T., Bouaouina, M., Elliott, P. R., Bate, N., Patel, B., Gingras, A. R., Grossmann, J.G., Roberts, G. C. K., Calderwood, D. A., Critchley, D. R., and Barsukov, I. L.(2010).Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation.The EMBO Journal29(6),1069-1080.
Grage, K., Jahns, A. C., Parlane, N., Palanisamy, R., Rasiah, I. A., Atwood, J. A., and Rehm,B. H. A.(2009). Bacterial polyhydroxyalkanoate granules: Biogenesis, structure, andpotential use as nano-/micro-beads in biotechnological and biomedical applications.Biomacromolecules10(4),660-669.
Hahn, J. J., Eschenlauer, A. C., Sleytr, U. B., Somers, D. A., and Srienc, F.(1999).Peroxisomes as sites for synthesis of polyhydroxyalkanoates in transgenic plants.Biotechnology Progress15(6),1053-1057.
Hamada, H., Arakawa, T., and Shiraki, K.(2009). Effect of additives on protein aggregation.Current Pharmaceutical Biotechnology10(4),400-407.
Han, M.-J., Lee, J. W., Lee, S. Y., and Yoo, J. S.(2008). Proteome-Level Responses ofEscherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter inProtein Production. Journal of Biomedicine and Biotechnology2008,1-12.
Hansen, M., Hsu, A.-L., Dillin, A., and Kenyon, C.(2005). New Genes Tied to Endocrine,Metabolic, and Dietary Regulation of Lifespan from a Caenorhabditis elegans GenomicRNAi Screen. PLoS Genetics1(1), e17.
Hennig, S., Strauss, H. M., Vanselow, K., Yildiz,., Schulze, S., and Arens, J.(2009).Structural and functional analyses of PAS domain interactions of the clock proteinsDrosophila PERIOD and mouse PERIOD2. Plos Biology7, e1000094.
Hiroyasu, S., Takashi, M., and Tomonori, M.(1999). PHA production by activated sludge.International Journal of Biological Macromolecules25,105-109.
Hisano, T., Tsuge, T., Fukui T., Iwata, T., Miki, K., and Doi, Y.(2003). Crystal structure of the(R)-specific enoyl-CoA hydratase from Aeromonas caviae involved inpolyhydroxyalkanotes biosynthesis. Journal of Biological Chemistry278,617-624.
Hoffmann, N., Amara, A. A., Beermann, B. B., Qi, Q., Hinz, H. J., and Rehm, B. H.(2002).Biochemical characterization of the pseudomonas putida3-hydroxyacyl ACP: CoAtransacylase, which diverts intermediates of fatty acid de novo biosynthesis. Journal ofBiological Chemistry277(45),42926-42936.
Hoffmann, N., Steinbüchel, A., and Rehm, B. H. A.(2000). The Pseudomonas aeruginosaphaG gene product is involved in the synthesis of polyhydroxyalkanoic acid consistingof medium-chain-length constituents from non-related carbon sources. FEMSMicrobiology Letters184,253-259.
Holm, L., and Sander, C.(1994). Searching protein structure databases has come of age.Proteins19,165-173.
Houmiel, K. L., Slater, S., Broyles, D., Casagrande, L., Colburn, S., Gonzalez, K., Mitsky, T.A., Reiser, S. E., Shah, R. D., Taylor, N. B., Tran, M., Valentin, H. E., and Gruys, K. J.(1999). Poly-(β-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus.Planta209,547-550.
Huang, Z., Feng, Y., Chen, D., Wu, X., Huang, S., Wang, X., Xiao, X., Li, W., Huang, N., Gu,L., Zhong, G., and Chai, J.(2008). Structural basis for activation and inhibition of thesecreted Chlamydia protease CPAF. Cell Host Microbe4,529-542.
Hubbard, P. A., Yu, W., Schulz, H., and Kim, J.-J. P.(2009). Domain swapping in thelow-similarity isomerase/hydratase superfamily: The crystal structure of ratmitochondrial Δ3, Δ2-enoyl-CoA isomerase. Protein Science14(6),1545-1555.
Hurtado-Gonzaleso., Aragon-Caballero L. and Apaza-Tapia W.(2008). Survival and Spread ofPhytophthora capsici in Coastal Peru. Phytopathology98(6),688-694.
Ito, L., Kobayashi, T., Shiraki, K. and Yamaguchi, H.(2008). Effect of amino acids and aminoacid derivatives on crystallization of hemoglobin and ribonuclease A. Journal ofSynchrotron Radiation15(3),316-318.
Jackson, K. L., Yin, J., Csinos, A. S., and Ji, P.(2010). Fungicidal activity of fluopicolide forsuppression of Phytophthora capsici on squash. Crop Protection29(12),1421-1427.
Jae Park, S., and Yup Lee, S.(2004). New fadB homologous enzymes and their use inenhanced biosynthesis of medium-chain-length polyhydroxyalkanoates infadBmutantEscherichia coli. Biotechnology and Bioengineering86(6),681-686.
Jancarik, J., and Kim, S. H.(1991). Sparse-matrix sampling-a screening method forcrystallization of proteins. Journal of applied crystallography24,409-411.
Jandrossek, D., Schirmer, A., and Schlegel, H. G.(1996). Biodegradation ofpolyhydroxyalkanoic acids. Applied Microbiology and Biotechnology46,451-463.
Jiang, Y., Hebly, M., Kleerebezem, R., Muyzer, G., and van Loosdrecht, M. C. M.(2011).Metabolic modeling of mixed substrate uptake for polyhydroxyalkanoate (PHA)production. Water Research45(3),1309-1321.
Jimena, A. R., Nancy, I. L., Ruben, O. F., and Beatriz, S. M.(2001). Polyhydroxyalkanoatedegradation is associated with nucleotide accumulation and enhances stress resistanceand survival of Pseudomonas oleovorans in natural water microcosms. Applied andEnvironmental Microbiology67(1),225-230.
John, M. E., and Keller, G.(1996). Metabolic pathway engineering in cotton: Biosynthesis ofpolyhydroxybutyrate in fiber cells. Proceedings of the National Academy of Sciences ofUSA93,12768-12773.
Kartha, G., and Parthasarathy, R.(1965). Combination of multiple isomorphous replacementand anomalous dispersion data for protein structure determination. Ⅰ. Determination ofheavy-atom positions in protein derivatives. Acta Crystallographica18(4),745-749.
Kartha, G., and Parthasarathy, R.(1965). Combination of multiple isomorphous replacementand anomalous dispersion data for protein structure determination. Ⅱ. Correlation of theheavy-atom positions in different isomorphous protein crystals. Acta Crystallographica18(4),749-753.
Kartha, G.(1965). Combination of multiple isomorphous replacement and anomalousdispersion data for protein structure determination.3. Refinement of heavy atompositions by the least-squares method. Acta Crystallographica19(6),883-885.
Kawashima, Y., Cheng, W., Mifune, J., Orita, I., Nakamura, S., and Fukui, T.(2011).Characterization and Functional Analyses of R-Specific Enoyl Coenzyme A Hydratasesin Polyhydroxyalkanoate-Producing Ralstonia eutropha. Applied and EnvironmentalMicrobiology78(2),493-502.
Khanna, S., and Srivastava, A. K.(2005). Recent advances in microbialpolyhydroxyalkanoates. Process Biochemistry40(2),607-619.
Koski, M. K.(2004). A Two-domain Structure of One Subunit Explains Unique Features ofEukaryotic Hydratase2. Journal of Biological Chemistry279(23),24666-24672.
Kristian Koski, M., Haapalainen, A. M., Hiltunen, J. K., and Glumoff, T.(2005). CrystalStructure of2-Enoyl-CoA Hydratase2from Human Peroxisomal MultifunctionalEnzyme Type2. Journal of Molecular Biology345(5),1157-1169.
Langenbach, S., Rehm, B. H. A., and Steinbüchel, A.(1997). Functional expression of thePHA synthase gene phaC1from Pseudomonas aeruginosa in Escherichia coli results inpoly(3-hydroxyalkanoate) synthesis. FEMS Microbiology Letters150,303-309.
Laskowski, R. A., Moss, D. S., and Thornton, J. M.(1993). Main-chain bond lengths andbond angles in protein structures. Journal of Molecular Biology231,1049-1067.
Leaversuch, R.(1987). Industry weighs the need to make polymer degradable. ModernPlastics64,52-55.
Leduc, D., Battesti, A., and Bouveret, E.(2007). The Hotdog Thioesterase EntH (YbdB) Playsa Role In Vivo in Optimal Enterobactin Biosynthesis by Interacting with the ArCPDomain of EntB. Journal of Bacteriology189(19),7112-7126.
Leesong, M., Henderson, B. S., Gillig, J. R., Schwab, J. M., and Smith, J. L.(1996). Structureof a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturatedfatty acids: two catalytic activities in one active site. Structure4,253-264.
Lee, S. Y., and Chang, H. N.(1995). Production of poly-(hydroxyalkanoic acid). Advances inBiochemical Engineering/Biotechnology52,27-58.
Lee, S. Y., and Choi, J.(1999). Production and degradation of polyhydroxyalkanoates inwaste environment. Waste Management19,133-139.
Lee, S. Y.(1996). Bacterial polyhydroxyalkanoates. Biotechnology and Bioengineering49,1-14.
Lee, S. Y., Park, S. H., Lee, Y., and Lee, S. H.(2001). Production of chiral and other valuablecompounds from microbial polyesters. In: Doi, Y., Steinbüchel, A.(eds.), BiopolymersWiley-VCH, Weinheim, Germany375-88.
Leslie, A. G. W.(2006). The integration of macromolecular diffraction data. ActaCrystallographica Section D Biological Crystallography62,48-57.
Li, J., Derewenda, U., Dauter, Z., Smith, S., and Derewenda, Z. S.(2000). Crystal structure ofthe Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme.Nature Structural&Molecular Biology7,555-559.
Li, R., Zhang, H., and Qi, Q.(2007). The production of polyhydroxyalkanoates inrecombinant Escherichia coli. Bioresource Technology98(12),2313-2320.
Li, Y., Zhang, Z., Robinson, G. E., and Palli, S. R.(2007). Identification and Characterizationof a Juvenile Hormone Response Element and Its Binding Proteins. Journal ofBiological Chemistry282(52),37605-37617.
Lu, Q. H., Han, J., Zhou, L. G., Zhou, J., and Xiang, H.(2008). Genetic and biochemicalcharacterization of the Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase inHaloferax mediterranei. Journal of Bacteriology190,4173-4180.
Madison, L. L., and Huisman, G. W.(1999). Metabolic engineering ofpoly(3-hydroxyalkanoates): from DNA to plastic. Microbiology and Molecular BiologyReviews63(1),21-53.
Main, P., and Rossmann, M. G.(1966). Relationship among structure factors due to identicalmolecules in different crystallographic environments. Acta Crystallographica21(1),67-72.
Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sanchez, R., Melo, F., and Sali, A.(2000).Comparative protein structure modeling of genes and genomes. Annual Review ofBiophysics and Biomolecular Structure29,291-325.
Mathieu, M., Zeelen, J. P., Pauptit, R. A., Erdmann, R., Kunau, W. H., and Wierenga, R. K.(1994). The2.8crystal structure of peroxisomal3-ketoacyl-CoA thiolase ofSaccharomyces cerevisiae: a five-layered αβαβα structure constructed from two coredomains of identical topology. Structure2(9),797-808.
Matthews, B. W.(1968). Solvent content of protein crystals. Journal of Molecular Biology33,491-497.
Morris, R. J., Perrakis, A., and Lamzin, V. S.(2003). ARP/wARP and automatic interpretationof protein electron density maps. Methods in Enzymology374,229-244.
Mothes, G., Babel, W.(1995). Methylobacterium rhodesianum MB126possesses twostereospecific crotonyl-CoA hydratase. Canadian Journal of Microbiology41,68-72.
Motin, V. L., Georgescu, A. M., Elliott, J. M., Hu, P., Worsham, P. L., Ott, L. L., Slezak, T. R.,Sokhansanj, B. A., Regala, W. M., Brubaker, R. R., and Garcia, E.(2002). GeneticVariability of Yersinia pestis Isolates as Predicted by PCR-Based IS100Genotyping andAnalysis of Structural Genes Encoding Glycerol-3-Phosphate Dehydrogenase (glpD).Journal of Bacteriology184(4),1019-1027.
Murshudov, G. N., Vagin, A. A., and Dodson, E. J.(1997). Refinement of macromolecularstructures by the maximum-likelihood method. Acta Crystallographica Section DBiological Crystallography53(3),240-255.
Nanev, C. N.(2007). On the slow kinetics of protein crystallization. Crystal Growth&Design7(8),1533-1540.
Nawrath, C., Poirier, Y., and Somerville, C.(1994). Targeting of the polyhydroxybutyratebiosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels ofpolymer accumulation. Proceedings of the National Academy of Sciences of USA91,12760-12764.
Ozaki, S., and Katayama, T.(2009). DnaA structure, function, and dynamics in the initiationat the chromosomal origin. Plasmid62(2),71-82.
Park, S. J., Ahn, W. S., Green, P. R., and Lee, S. Y.(2001). Production ofpoly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by metabolically engineeredEscherichia coli strains. Biomacromolecules2,248-254.
Park, S. J., and Lee, S. Y.(2003). Identification and Characterization of a New EnoylCoenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-LengthPolyhydroxyalkanoates in Recombinant Escherichia coli. Journal of Bacteriology185(18),5391-5397.
Park, S. J., Park, J. P., and Lee, S. Y.(2002). Metabolic engineering of Escherichia coli for theproduction of medium-chain-length polyhydroxyalkanoates rich in specific monomers.FEMS Microbiology Letters214,217-222.
Perrin, M. H., Grace, C. R. R., DiGruccio, M. R., Fischer, W. H., Maji, S. K., Cantle, J. P.,Smith, S., Manning, G., Vale, W. W., and Riek, R.(2007). Distinct Structural andFunctional Roles of Conserved Residues in the First Extracellular Domain of Receptorsfor Corticotropin-releasing Factor and Related G-protein-coupled Receptors. Journal ofBiological Chemistry282(52),37529-37536.
Phan, I., Subramanian, S., Olsen, C., Edwards, T. E., Guo, W., Zhang, Y., Van Voorhis, W. C.,Stewart, L. J., and Myler, P. J.(2011). Structure of fumarate hydratase fromRickettsiaprowazekii, the agent of typhus and suspected relative of the mitochondria. ActaCrystallographica Section F Structural Biology and Crystallization Communications67(9),1123-1128.
Pidugu, L. S., Maity, K., Ramaswamy, K., Surolia, N., and Suguna, K.(2009). Analysis ofproteins with the “Hot dog” fold: Prediction of function and identification of catalyticresidues of hypothetical proteins. BMC Structural Biology9(1),1-16.
Poirier, Y., Dennis, D. E., Klomparens, K., and Somerville, C.(1992). Polyhydroxybutyrate, abiodegradable thermoplastic, produced in transgenic plants. Science256,520-523.
Putnam, N. H., Srivastava, M., Hellsten, U., Dirks, B., Chapman, J., Salamov, A., Terry, A.,Shapiro, H., Lindquist, E., Kapitonov, V. V., Jurka, J., Genikhovich, G., Grigoriev, I. V.,Lucas, S. M., Steele, R. E., Finnerty, J. R., Technau, U., Martindale, M. Q., and Rokhsar,D. S.(2007). Sea Anemone Genome Reveals Ancestral Eumetazoan Gene Repertoire andGenomic Organization. Science317(5834),86-94.
Qin, Y. M., Haapalainen, A. M., Kilpel inen, S. H., Marttila, M. S., Koski, M. K., Glumoff, T.,Novikov, D. K., and Hiltunen, J. K.(2000). Human peroxisomal multifunctional enzymetype2. Site-directed mutagenesis studies show the importance of two protic residues for2-enoyl-CoA hydratase2activity. Journal of Biological Chemistry275,4965-4972.
Qi, Q., Rehm, B. H. A., and Steinbüchel, A.(1997). Synthesis of poly(3-hydroxyalkanoates)in Escherichia coli expressing the PHA synthase gene phaC2from Pseudomonasaeruginosa: comparison of PhaC1and PhaC2. FEMS Microbiology Letters157,155-162.
Qi, Q., Steinbüchel, A., and Rehm, B. H. A.(1998). Metabolic routing towardspolyhydroxyalkanoic acid synthesis in recombinant Escherichia coli (fadR): inhibition offatty acid beta-oxidation by acrylic acid. FEMS Microbiology Letters167,89-94.
Otwinowski, Z., and Minor, W.(1997). Processing of X-ray diffraction data collected inoscillation mode. Methods in Enzymology276,307-326.
Rayment, I.(1997). Reductive alkylation of lysine residues to alter crystallization propertiesof proteins. Methods in Enzymology276,171-179.
Read, R. J.(2001). Pushing the boundaries of molecular replacement with maximumlikelihood. Acta Crystallographica Section D Biological Crystallography57(10),1373-1382.
Rehm, B. H. A., Kriiger, N., and Steinbuchel, A.(1998). A new metabolic link between fattyacid de novo synthesis and polyhydroxyalkanoic acid synthesis. The Journal ofBiological Chemistry273,24044-24051.
Rehm, B. H. A.(2003). Polyester synthases: natural catalysts for plastics. BiochemicalJournal376,15-33.
Rehm, B. H.(2007). Biogenesis of microbial polyhydroxyalkanoate granules: a platformtechnology for the production of tailor-made bioparticles. Current Issues in MolecularBiology9(1),41-62.
Reiser, S. E., Mitsky, T. A., and Gruys, K. J.(2000). Characterization and cloning of an(R)-specific trans-2,3-enoylacyl-CoA hydratase from Rhodospirillum rubrum and use ofthis enzyme for PHA production in Escherichia coli. Applied Microbiology andBiotechnology53,209-218.
Ren, Q., Sierro, N., Witholt, B., and Kessler, B.(2000). FabG, an NADPHdependent3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors formedium-chain-length poly-3-hydroxyalkanoate biosynthesis in Escherichia coli. Journalof Bacteriology182,2978-2981.
Robert, L., Robert, A. M., and Jacotot, B.(1998). Elastin elastase atherosclerosis revisited.Atherosclerosis140,281-295.
Robles-Yerena, L., Rodríguez-Villarreal, R. A., Ortega-Amaro, M. A., Fraire-Velázquez, S.,Simpson, J., Rodríguez-Guerra, R., and Jiménez-Bremont, J. F.(2010). Characterizationof a new fungal antagonist of Phytophthora capsici. Scientia Horticulturae125(3),248-255.
Rossier, O. M., Gauthier, N., Biais, N., Vonnegut, W., Fardin, M.-A., Avigan, P., Heller, E. R.,Mathur, A., Ghassemi, S., Koeckert, M. S., Hone, J. C., and Sheetz, M. P.(2010). Forcegenerated by actomyosin contraction builds bridges between adhesive contacts. TheEMBO Journal29(6),1055-1068.
Rossmann, M. G., Blow, D. M., Harding, M. M., and Coller, E.(1964) The relative positionsof independent molecules within the same asymmetric unit. Acta Crystallographica17(4),338-342.
Sacco, E., Legendre, V., Laval, F., Zerbib, D., Montrozier, H., Eynard, N., Guilhot, C., Daffé,M., and Quémard, A.(2007). Rv3389C from Mycobacterium tuberculosis, a member ofthe (R)-specific hydratase/dehydratase family. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics1774(2),303-311.
Saridakis, E., and Chayen, N. E.(2009). Towards a “universal” nucleant for proteincrystallization. Trends in Biotechnology27(2),99-106.
Schena, L., and Cooke, D. E. L.(2006). Assessing the potential of regions of the nuclear andmitochondrial genome to develop a “molecular tool box” for the detection andcharacterization of Phytophthora species. Journal of Microbiological Methods67(1),70-85.
Scibetta, S., Schena, L., Chimento, A., Cacciola, S. O., and Cooke, D. E. L.(2012). Amolecular method to assess Phytophthora diversity in environmental samples. Journal ofMicrobiological Methods88(3),356-368.
Shandilya, S. M. D., Nalam, M. N. L., Nalivaika, E. A., Gross, P. J., Valesano, J. C., Shindo,K., Li, M., Munson, M., Royer, W. E., Harjes, E., Kono, T., Matsuo, H., Harris, R. S.,Somasundaran, M., and Schiffer, C. A.(2010). Crystal Structure of the APOBEC3GCatalytic Domain Reveals Potential Oligomerization Interfaces. Structure18(1),28-38.
Shi, R., Proteau, A., Villarroya, M., Moukadiri, I., Zhang, L. H., Trempe, J. F., Matte, A.,Armengod, M. E., and Cygler, M.(2010). Structural basis for Fe-S cluster assembly andtRNA thiolation mediated by IscS protein-protein interactions. Plos Biology8,e1000354.
Sholberg P. L., Walkerm C. O. and Gorman D. T.(2007). First Report of Phytophthora capsicion Cucurbits and Peppers in British Columbia. Canadian Journal of Plant Pathology29(2),153-158.
Shrivastav, A., Mishra, S. K., and Mishra, S.(2010). Polyhydroxyalkanoate (PHA) synthesisby Spirulina subsalsa from Gujarat coast of India. International Journal of BiologicalMacromolecules46(2),255-260.
Silke, F., Alexander, S., and Bernd, H. A.(2000). PhaG-mediated synthesis of poly-3-hydroxyalkanoates consisting of medium-chain–length constituents from nonrelatedcarbon sources in recombinant pseudomonas fragi. Applied and EnvironmentalMicrobiology66(5),2117-2124.
Studholme, D. J., and Dixon, R.(2003). Domain Architectures of54-DependentTranscriptional Activators. Journal of Bacteriology185(6),1757-1767.
Sundaramoorthy, R., Micossi, E., Alphey, M. S., Germain, V., Bryce, J. H., Smith, S. M.,Leonard, G. A., and Hunter, W. N.(2006). The crystal structure of a plant3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acidbeta-oxidation. Journal of Molecular Biology359(2),347-357.
Taguchi, K., Aoyagi, Y., Matsusaki, H., Fukui, T., and Doi, Y.(1999). Co-expression of3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHAproduction in Escherichia coli HB101strain. FEMS Microbiology Letters176,183-190.
Taguchi, S., Nakamura, H., Kichise, T., Tsuge, T., Yamato, I., and Doi, Y.(2003). Productionof polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinantRalstonia eutropha using mutants of original PHA synthase. Biochemical EngineeringJournal16(2),107-113.
Takayama, K., Wang, C., and Besra, G. S.(2005). Pathway to Synthesis and Processing ofMycolic Acids in Mycobacterium tuberculosis. Clinical Microbiology Reviews18(1),81-101.
Takehara, M., Ino, K., Takakusagi, Y., Oshikane, H., Nureki, O., Ebina, T., Mizukami, F., andSakaguchi, K.(2008). Use of layer silicate for protein crystallization: effects ofmicromica and chlorite powders in hanging drops. Analytical Biochemistry373(2),322-329.
Taylor, G.(2003). The phase problem. Acta Crystallographica Section D BiologicalCrystallography59(11),1881-1890.
Thakur, A. S., Robin, G., Guncar, G., Saunders, N. F. W., Newman, J., Martin, J. L., and Kobe,B.(2007). Improved success of sparse matrix protein crystallization screening withheterogeneous nucleating agents. Plos one2(10),1091.
Tian, X., Wang, A., Qiao, S., Zhang, G., Xi, J., Li, X., Liu, Y., and Xu, Y.(2009). Expression,purification and characterization of a functional extracellular domain of porcine FcγRII.Protein Expression and Purification68(1),12-17.
Tmaietti G. and Valentino D.(2001). Physiological Characterisation of a Population ofPhytophthora capsici Leon.from Northern Italy. Journal of Plant Pathology83(3),199-205.
Tollin, P., and Rossmann, M. G.(1966). A description of various rotation function programs.Acta Crystallographica21(6),872-876.
Tollin, P., Main, P., and Rossmann, M. G.(1966). The symmetry of the rotation function. ActaCrystallographica20(3),404-407.
Truong, N. V., Liew, E. C. Y., and Burgess, L. W.(2010). Characterisation of Phytophthoracapsici isolates from black pepper in Vietnam. Fungal Biology114(2-3),160-170.
Tsuchida, S., Kawamoto, K., Nunome, K., Hamaue, N., Hui, S. P., Murai, T., Aoki, Takashi.,and Kurosawa, T.(2011). Chiral Separation, Determination of Absolute Configuration,and High-performance Liquid Chromatography Detection of Enantiomeric3-hydroxyhexadecanoyl-CoA. Journal of Oleo Science60(2),87-92.
Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A., and Doi Y.(1999).Molecular cloning of two (R)-specific enoyl-CoA Hydratase genes from Pseudomonasaeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS MicrobiologyLetters184,193-198.
Tsuge, T., Hisano, T., Taguchi, S., and Doi, Y.(2003). Alteration of Chain Length SubstrateSpecificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratasethrough Site-Directed Mutagenesis. Applied and Environmental Microbiology69(8),4830-4836.
Tsuge, T., Taguchi, K., Taguchi, S., and Doi, Y.(2003). Molecular characterization andproperties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa:metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid β-oxidation.International Journal of Biological Macromolecules31,195-205.
Tyler, B. M., Tripathy, S., Zhang, X., Dehal, P., Jiang, R. H., Aerts, A., Arredondo, F. D.,Baxter, L., Bensasson, D., Beynon, J. L., Chapman, J., Damasceno, C. M., Dorrance, A.E., Dou, D., Dickerman, A. W., Dubchak, I. L., Garbelotto, M., Gijzen, M., Gordon, S.G., Govers, F., Grunwald, N. J., Huang, W., Ivors, K. L., Jones, R. W., Kamoun, S.,Krampis, K., Lamour, K. H., Lee, M. K., McDonald, W. H., Medina, M., Meijer, H. J.,Nordberg, E. K., Maclean, D. J., Ospina-Giraldo, M. D., Morris, P. F., Phuntumart, V.,Putnam, N. H., Rash, S., Rose, J. K., Sakihama, Y., Salamov, A. A., Savidor, A.,Scheuring, C. F., Smith, B. M., Sobral, B. W., Terry, A., Torto-Alalibo, T. A., Win, J., Xu,Z., Zhang, H., Grigoriev, I. V., Rokhsar, D. S., Boore, J. L.(2006). Phytophthoragenome sequences uncover evolutionary origins and mechanisms of pathogenesis.Science313(5791),1261-1266.
Uta, B., Yaroslav, T., Gotthard, K., and Wolfgang, B.(2002). Yeasts as producers ofpolyhydroxyalkanoates: Genetic engineering of Saccharomyces cerevisiae.Macromolecular Bioscience2(8),380-386.
Vagin, A., and Teplyakov, A.(1997). MOLREP: an automated program for molecularreplacement. Journal of Applied Crystallography30,1022-1025.
Valappil, S. P., Peiris, D., Langley, G. J., Herniman, J. M., Boccaccini, A. R., Bucke, C., andRoy, I.(2007). Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelatedcarbon sources by a newly characterized Bacillus spp. Journal of Biotechnology127(3),475-487.
Venkata Mohan, S., and Venkateswar Reddy, M.(2013). Optimization of critical factors toenhance polyhydroxyalkanoates (PHA) synthesis by mixed culture using Taguchi designof experimental methodology. Bioresource Technology128,409-416.
Vivian, J. P., Riedmaier, P., Ge, H., Le Nours, J., Sansom, F. M., Wilce, M. C. J., Byres, E.,Dias, M., Schmidberger, J. W., Cowan, P. J., d'Apice, A. J. F., Hartland, E. L., Rossjohn,J., and Beddoe, T.(2010). Crystal Structure of a Legionella pneumophila Ecto-Triphosphate Diphosphohydrolase, A Structural and Functional Homolog of theEukaryotic NTPDases. Structure18(2),228-238.
Voinova, O. N., Gladyshev, M. I., and Volova, T. G.(2008). Comparative study of PHAdegradation in natural reservoirs having various types of ecosystems. MacromolecularSymposia269,34-37.
Volova, T. G., Boyandin, A. N., Vasiliev, A. D., Karpov, V. A., Prudnikova, S. V., Mishukova,O. V., Boyarskikh, U. A., Filipenko, M. L., Rudnev, V. P., Bá Xuan, B., Vi t D ng, V.,and Gitelson, I. I.(2010). Biodegradation of polyhydroxyalkanoates (PHAs) in tropicalcoastal waters and identification of PHA-degrading bacteria. Polymer Degradation andStability95(12),2350-2359.
Volova, T. G., Gladyshev, M. I., Trusova, M. Y., and Zhila, N. O.(2006). Degradation ofpolyhydroxyalkanoates and the composition of microbial destructors under naturalconditions. Microbiology75(5),593-598.
Volova, T. G., Gladyshev, M. I., Trusova, M. Y., and Zhila, N. O.(2007). Degradation ofpolyhydroxyalkanoates in eutrophic reservoir. Polymer Degradation and Stability92,580-586.
Vo, M. T., Lee, K.-W., Jung, Y.-M., and Lee, Y.-H.(2008). Comparative effect ofoverexpressed phaJ and fabG genes supplementing (R)-3-hydroxyalkanoate monomerunits on biosynthesis of mcl-polyhydroxyalkanoate in Pseudomonas putida KCTC1639.Journal of Bioscience and Bioengineering106(1),95-98.
Walter, T. S., Meier, C., Assenberg, R., Au, K. F., Ren, J., Verma, A., Nettleship, J. E., Owens,R. J., Stuart, D. I., and Grimes, J. M.(2006). Lysine methylation as a routine rescuestrategy for protein crystallization. Structure14(11),1617-1622.
Wang, Q., Xia, Y., Chen, Q., and Qi, Q.(2012). Incremental truncation of PHA synthasesresults in altered product specificity. Enzyme and Microbial Technology50(6-7),293-297.
Winn, M. D., Murshudov, G. N., and Papiz, M. Z.(2003). Macromolecular TLS refinement inREFMAC at moderate resolutions. Methods in Enzymology374,300-321.
Wu, L., Wang, L., Hua, G., Liu, K., Yang, X., Zhai, Y., Bartlam, M., Sun, F., and Fan, Z.(2009). Structural Basis for Proteolytic Specificity of the Human Apoptosis-InducingGranzyme M. The Journal of Immunology183(1),421-429.
Xu, H., Shi, Y., Wang, J., Jones, D., Weilrauch, D., Ying, R., Wakim, B., and Pritchard, K. A.(2007). A Heat Shock Protein90Binding Domain in Endothelial Nitric-oxide SynthaseInfluences Enzyme Function. Journal of Biological Chemistry282(52),37567-37574.
Xu, J., Zhao, X., Wang, X., Zhao, Z., and Du, Y.(2007). Oligochitosan inhibits Phytophthoracapsici by penetrating the cell membrane and putative binding to intracellular targets.Pesticide Biochemistry and Physiology88(2),167-175.
Yasui, N., Nogi, T., and Takagi, J.(2010). Structural Basis for Specific Recognition of Reelinby Its Receptors. Structure18(3),320-331.
You, Z., Omura, S., Ikeda, H., Cane, D. E., and Jogl, G.(2007). Crystal Structure of theNon-heme Iron Dioxygenase PtlH in Pentalenolactone Biosynthesis. Journal ofBiological Chemistry282(50),36552-36560.
Yücel S.(1995). A study on soil solarization and combined with fumigant application tocontrol Phytophthora crown blight (Phytophthora capsici Leonian) on peppers in theEast Mediterranean region of Turkey. Crop Protection14(8),653-655.
Zarzycki, J., Schlichting, A., Strychalsky, N., Muller, M., Alber, B. E., and Fuchs, G.(2007).Mesaconyl-Coenzyme A Hydratase, a New Enzyme of Two Central Carbon Metabolic
Pathways in Bacteria. Journal of Bacteriology190(4),1366-1374.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |