嗜热脂肪酶NC-loop对催化活性、稳定性和趋异进化的作用
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摘要
Loop作为蛋白质二级结构的重要组成元件，其柔性和运动性对于蛋白质功能，尤其是酶催化作用具有重要作用，如定位重要的催化残基、参与底物结合以及调控底物的进入和产物的释放。三级结构分析揭示α/β水解酶超家族成员（脂肪酶第五家族、环氧化物水解酶、卤代烷脱卤酶和过水解酶等家族）存在一段loop区（NC-loop）负责连接α/β水解酶催化结构域的N-末端和盖子结构域（Cap），并且NC-loop在序列、长度、柔性以及构象上差异很大。本论文以来源于嗜热细菌长白山闪烁杆菌（Fervidobacteriumchangbaicum）且属于脂肪酶第五家族的嗜热脂肪酶FClip1作为研究对象，利用蛋白质工程手段系统地研究了NC-loop在催化过程、稳定性和趋异进化中的功能性作用。
首先，根据残基的溶剂可及性表面积（Solvent Accessible Surface），我们将FClip1的NC-loop（Asp131-Lys151）分为N-末端（酶分子内部区域，Asp131-Ser138）和C-末端（酶分子表面区域，Glu139-Lys151）两部分，并设计和构建了N-末端和C-末端不同长度的系列截短突变体；结果表明删除N-末端的3-8个残基均导致酶的催化活性显著降低（对对硝基苯酚丁酸酯的活力降低67-500倍），说明N-末端对于酶的催化作用至关重要；而删除C-末端的3-13个残基促进了酶催化活性的提高（野生型的125-221%）、78℃下热稳定性的提高（3.0-11.9倍）以及对盐酸胍半失活浓度的提高（野生型的105-167%），表明C-末端不参与酶的催化作用。其次，我们也使用丙氨酸扫描突变来确定N-末端对酶催化活性有重要影响的残基，然而活性测定表明丙氨酸的替换并未使酶的活性显著降低（野生型的71-223%），说明N-末端残基的侧链即使只保留一个甲基也不会损失酶的催化活性。进一步定点饱和突变分析显示保守残基Tyr134突变为Glu后，酶的催化活性降低为野生型4.5%，表明NC-loop对酶催化作用的重要性不仅体现在N-末端区域的构象完整性上，而且还与N-末端的氨基酸组成相关。最后，利用环化排列技术研究了C-末端区域的柔性和可塑性，突变体CP146和CP152仍保持较高的催化活性（野生型的28-52%），表明C-末端区域具有一定的柔性和可塑性；结合C-末端区域残基的删除提高了酶的活性和稳定性表明NC-loop的C-末端区域作为可塑区可以进行酶分子再设计而进一步优化酶学性质。
FClip1的NC-loop既有其他α/β水解酶NC-loop的共同点——参与底物口袋和底物通道入口的形成，也存在明显的差异——长度、序列、构象和功能不同，推测NC-loop在α/β水解酶超家族的趋异进化中起重要作用，此外，FClip1的NC-loop序列和长度与过水解酶家族的更相似，同时FClip1也表现出较低的过水解酶活性（3mU/mg），表明FClip1可能是脂肪酶和过水解酶家族间的进化中间体。
Loop was the important element of secondary structure. Flexible loops play importantroles in protein functions. The elegant catalytic mechanism of enzymes relies on mobility andflexibility of functional loops in many aspects, such as orientating important catalytic residues,participating in the substrate binding, and regulating the entrance of substrates and the releaseof products. It has been shown that the members of α/β hydrolase superfamily (lipase Vfamily, epoxide hydrolases, haloalkane dehalogenases, and perhydrolases) possessed theNC-loop connecting the N-terminal of the catalytic domain and the Cap domain. NC-loopswere diverse in the sequence, length, flexibility, and conformation. In this work, we took thethermostable lipase FClip1from Fervidobacterium changbaicum and belonging to lipase Vfamily as a research model, investigated systemically the role of NC-loop of the lipase FClip1on catalytic process, stability, and divergent evolution by protein engineering.
The model of FClip1possesses a catalytic domain and a cap domain. NC-loop contains21residues (Asp131-Lys151). Based on the solvent accessibility of residues, NC-loop can beroughly divided into two parts. The N-terminal (Asp131-Ser138) is located in the proteininterior and participates in the formation of the substrate binding pocket, while the C-terminal(Glu139-Lys151) is a highly surface-exposed region. We designed and constructed a series ofmutants by systematically truncating the NC-loop from its N-terminal or C-terminal.Deletions from the N-terminal or C-terminal part of NC-loop had different effects on activity.Deletions from the N-terminal caused the enzyme to retain no more than1.5%activity of wildtype. Removing as small as three residues could greatly inactivate the enzyme. TheN-terminal was very important for catalytic activity of the enzyme. However, deletions fromthe C-terminal improved the catalytic activity, the thermal stability at78oC, and the resistanceagainst GdnHCl. It was suggested that the C-terminal was not involved in the enzyme catalysis.
Ala-scanning mutagenesis on the N-terminal and saturation mutagenesis at the Tyr134were carried out to determine the important residues for enzyme catalysis. Enzyme assayexhibited that the introduction of alanine did not change the enzyme activity of FClip1. Thissuggested that the side chains of N-terminal residues retaining only a methyl group did notlose the catalytic activity of the enzyme. Besides, the enzymatic activity of the mutant Y134Ereduced about22-fold. It indicated that the importance of NC-loop for enzyme catalysis notonly reflected in the confomational integrity, but also was related with the amino acidcomposition. We also used circular permutation to probe the flexibility and plasticity ofNC-loop. The permutants CP146and CP152maintained high enzymatic activity. It showedthat the C-terminal of NC-loop was flexible and plastic, and might be a target region forenzyme redesign to optimize further the enzymatic properties.
The NC-loop of FClip1shared some common features with those of other α/β hydrolases,such as participating the formation of substrate pocket and the entrance of substrate channel.There are also obvious differences, such as diverse length, sequence, conformation, andfunction. This suggested that NC-loop might play an important role in the divergent evolutionof α/β hydrolase superfamily. In addition, the sequence and length of NC-loop of FClip1weremore similar with perhydrolases, and FClip1displayed a low activity of perhydrolysis (3mU/mg). It indicated that FClip1might be the evolutionary intermediate between lipases andperhydrolases.
首先，根据残基的溶剂可及性表面积（Solvent Accessible Surface），我们将FClip1的NC-loop（Asp131-Lys151）分为N-末端（酶分子内部区域，Asp131-Ser138）和C-末端（酶分子表面区域，Glu139-Lys151）两部分，并设计和构建了N-末端和C-末端不同长度的系列截短突变体；结果表明删除N-末端的3-8个残基均导致酶的催化活性显著降低（对对硝基苯酚丁酸酯的活力降低67-500倍），说明N-末端对于酶的催化作用至关重要；而删除C-末端的3-13个残基促进了酶催化活性的提高（野生型的125-221%）、78℃下热稳定性的提高（3.0-11.9倍）以及对盐酸胍半失活浓度的提高（野生型的105-167%），表明C-末端不参与酶的催化作用。其次，我们也使用丙氨酸扫描突变来确定N-末端对酶催化活性有重要影响的残基，然而活性测定表明丙氨酸的替换并未使酶的活性显著降低（野生型的71-223%），说明N-末端残基的侧链即使只保留一个甲基也不会损失酶的催化活性。进一步定点饱和突变分析显示保守残基Tyr134突变为Glu后，酶的催化活性降低为野生型4.5%，表明NC-loop对酶催化作用的重要性不仅体现在N-末端区域的构象完整性上，而且还与N-末端的氨基酸组成相关。最后，利用环化排列技术研究了C-末端区域的柔性和可塑性，突变体CP146和CP152仍保持较高的催化活性（野生型的28-52%），表明C-末端区域具有一定的柔性和可塑性；结合C-末端区域残基的删除提高了酶的活性和稳定性表明NC-loop的C-末端区域作为可塑区可以进行酶分子再设计而进一步优化酶学性质。
FClip1的NC-loop既有其他α/β水解酶NC-loop的共同点——参与底物口袋和底物通道入口的形成，也存在明显的差异——长度、序列、构象和功能不同，推测NC-loop在α/β水解酶超家族的趋异进化中起重要作用，此外，FClip1的NC-loop序列和长度与过水解酶家族的更相似，同时FClip1也表现出较低的过水解酶活性（3mU/mg），表明FClip1可能是脂肪酶和过水解酶家族间的进化中间体。
Loop was the important element of secondary structure. Flexible loops play importantroles in protein functions. The elegant catalytic mechanism of enzymes relies on mobility andflexibility of functional loops in many aspects, such as orientating important catalytic residues,participating in the substrate binding, and regulating the entrance of substrates and the releaseof products. It has been shown that the members of α/β hydrolase superfamily (lipase Vfamily, epoxide hydrolases, haloalkane dehalogenases, and perhydrolases) possessed theNC-loop connecting the N-terminal of the catalytic domain and the Cap domain. NC-loopswere diverse in the sequence, length, flexibility, and conformation. In this work, we took thethermostable lipase FClip1from Fervidobacterium changbaicum and belonging to lipase Vfamily as a research model, investigated systemically the role of NC-loop of the lipase FClip1on catalytic process, stability, and divergent evolution by protein engineering.
The model of FClip1possesses a catalytic domain and a cap domain. NC-loop contains21residues (Asp131-Lys151). Based on the solvent accessibility of residues, NC-loop can beroughly divided into two parts. The N-terminal (Asp131-Ser138) is located in the proteininterior and participates in the formation of the substrate binding pocket, while the C-terminal(Glu139-Lys151) is a highly surface-exposed region. We designed and constructed a series ofmutants by systematically truncating the NC-loop from its N-terminal or C-terminal.Deletions from the N-terminal or C-terminal part of NC-loop had different effects on activity.Deletions from the N-terminal caused the enzyme to retain no more than1.5%activity of wildtype. Removing as small as three residues could greatly inactivate the enzyme. TheN-terminal was very important for catalytic activity of the enzyme. However, deletions fromthe C-terminal improved the catalytic activity, the thermal stability at78oC, and the resistanceagainst GdnHCl. It was suggested that the C-terminal was not involved in the enzyme catalysis.
Ala-scanning mutagenesis on the N-terminal and saturation mutagenesis at the Tyr134were carried out to determine the important residues for enzyme catalysis. Enzyme assayexhibited that the introduction of alanine did not change the enzyme activity of FClip1. Thissuggested that the side chains of N-terminal residues retaining only a methyl group did notlose the catalytic activity of the enzyme. Besides, the enzymatic activity of the mutant Y134Ereduced about22-fold. It indicated that the importance of NC-loop for enzyme catalysis notonly reflected in the confomational integrity, but also was related with the amino acidcomposition. We also used circular permutation to probe the flexibility and plasticity ofNC-loop. The permutants CP146and CP152maintained high enzymatic activity. It showedthat the C-terminal of NC-loop was flexible and plastic, and might be a target region forenzyme redesign to optimize further the enzymatic properties.
The NC-loop of FClip1shared some common features with those of other α/β hydrolases,such as participating the formation of substrate pocket and the entrance of substrate channel.There are also obvious differences, such as diverse length, sequence, conformation, andfunction. This suggested that NC-loop might play an important role in the divergent evolutionof α/β hydrolase superfamily. In addition, the sequence and length of NC-loop of FClip1weremore similar with perhydrolases, and FClip1displayed a low activity of perhydrolysis (3mU/mg). It indicated that FClip1might be the evolutionary intermediate between lipases andperhydrolases.
引文
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