肺炎克氏杆菌M5al菌株中编码分解代谢3-羟基苯甲酸酶系的基因簇功能的研究
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摘要
芳香烃的代谢主要有三条途径，即邻位裂解途径，间位裂解途径和龙胆酸途径。其中，龙胆酸途径在生物化学,尤其是分子生物学方面的研究甚少，是一条重要而又需要深入研究的代谢途径。
Klebsiella pneumoniae M5a1菌株能以3-羟基苯甲酸作为唯一碳源和能源生长, 3-羟基苯甲酸的代谢是通过龙胆酸途径进行的。编码代谢3-羟基苯甲酸至丙酮酸的基因被克隆至一8kb的DNA片段上(pBSI质粒), 含有该质粒的大肠杆菌获得了以3-羟基苯甲酸作为唯一碳源和能源生长的能力。本研究首次双向测定此8.21kb的片段序列，推测其包含8个ORF，再通过同源性分析，推断其中四个ORF有可能表达具有功能的蛋白。利用PCR扩增，分别克隆出这四个基因，并将其连接到表达载体上，最终在大肠杆菌Rosseta中都得到了高效的表达。对四个基因分别编码的酶，3-羟基苯甲酸-6-单加氧酶，龙胆酸1,2-双加氧酶，顺丁烯二酸-单酰丙酮酸异构酶，反丁烯二酸-单酰丙酮酸水解酶分别进行功能研究（酶活性分析），结果表明这些酶能在体外将3-羟基苯甲酸分别转化成龙胆酸, 顺丁烯二酸-单酰丙酮酸, 反丁烯二酸-单酰丙酮酸, 最后生成丙酮酸和反丁烯二酸。采用亲和层析法纯化了其中的3-羟基苯甲酸6-单加氧酶（MhbM），并对其进行了最适pH，最适温度，稳定性，对3-羟基苯甲酸取代物的生物催化等性质的研究。采用移码突变的方法鉴定了该片段上另外二个基因mhbR（转录调节基因）和mhbT（底物转运基因）的功能。
此研究为国家自然科学基金项目(No. 30170036)资助，该课题是第二例在分子生物学水平上研究了整个龙胆酸途径, 而且其中的3-羟基苯甲酸-6-单加氧酶是第一次在分子生物学水平上得到研究。
Gentisate (2,5-dihydroxybenzoate) pathway is one of the three typical routes for bacterial aromatic catabolism. This pathway plays an important role in bacterial aromatic catabolism, but little is known at the biochemical and molecular levels compared with the other two pathways through catechol metabolism.
Klebsiella pneumoniae M5a1 metabolizes 3-hydroxybenzoate via gentisate to central metabolites. We have determined the sequence of a 8kb region spanning the mhb genes. The individual genes from the cluster mhbRTMDIH of 3-hydroxybenzoate-degrading Klebsiella pneumoniae M5a1 were cloned and overexpressed. By cloning into expression vectors and by biochemical analyses, four of these genes (mhbMDIH) have been shown to encode the enzymes involved in the catabolism of 3-hydroxybenzoate to fumarate and pyruvate through the gentisate pathway. MhbM is a 3-hydroxybenzoate-6-hydroxylase that catalyzed the para-hydroxylation of 3-hydroxybenzoate(3-HBA) to 2,5-dihydroxybenzoate (gentisate). MhbD is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate. MhbI is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. MhbH is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. MhbM is also purified and its dynamics characterization was performed. Frame-shift mutants of mhbR and mhbT were also made to investigate their possible roles in 3-hydroxybenzoate catabolism.
This project is funded by the National Natural Science Foundation of China (No. 30170036). The current result reported here is the second example of the research on
the genes encoding enzymes involved in the entire gentisate pathway and the 3-hydroxybenzoate-6-hydroxylase was studied at molecular level for the first time.
Klebsiella pneumoniae M5a1菌株能以3-羟基苯甲酸作为唯一碳源和能源生长, 3-羟基苯甲酸的代谢是通过龙胆酸途径进行的。编码代谢3-羟基苯甲酸至丙酮酸的基因被克隆至一8kb的DNA片段上(pBSI质粒), 含有该质粒的大肠杆菌获得了以3-羟基苯甲酸作为唯一碳源和能源生长的能力。本研究首次双向测定此8.21kb的片段序列，推测其包含8个ORF，再通过同源性分析，推断其中四个ORF有可能表达具有功能的蛋白。利用PCR扩增，分别克隆出这四个基因，并将其连接到表达载体上，最终在大肠杆菌Rosseta中都得到了高效的表达。对四个基因分别编码的酶，3-羟基苯甲酸-6-单加氧酶，龙胆酸1,2-双加氧酶，顺丁烯二酸-单酰丙酮酸异构酶，反丁烯二酸-单酰丙酮酸水解酶分别进行功能研究（酶活性分析），结果表明这些酶能在体外将3-羟基苯甲酸分别转化成龙胆酸, 顺丁烯二酸-单酰丙酮酸, 反丁烯二酸-单酰丙酮酸, 最后生成丙酮酸和反丁烯二酸。采用亲和层析法纯化了其中的3-羟基苯甲酸6-单加氧酶（MhbM），并对其进行了最适pH，最适温度，稳定性，对3-羟基苯甲酸取代物的生物催化等性质的研究。采用移码突变的方法鉴定了该片段上另外二个基因mhbR（转录调节基因）和mhbT（底物转运基因）的功能。
此研究为国家自然科学基金项目(No. 30170036)资助，该课题是第二例在分子生物学水平上研究了整个龙胆酸途径, 而且其中的3-羟基苯甲酸-6-单加氧酶是第一次在分子生物学水平上得到研究。
Gentisate (2,5-dihydroxybenzoate) pathway is one of the three typical routes for bacterial aromatic catabolism. This pathway plays an important role in bacterial aromatic catabolism, but little is known at the biochemical and molecular levels compared with the other two pathways through catechol metabolism.
Klebsiella pneumoniae M5a1 metabolizes 3-hydroxybenzoate via gentisate to central metabolites. We have determined the sequence of a 8kb region spanning the mhb genes. The individual genes from the cluster mhbRTMDIH of 3-hydroxybenzoate-degrading Klebsiella pneumoniae M5a1 were cloned and overexpressed. By cloning into expression vectors and by biochemical analyses, four of these genes (mhbMDIH) have been shown to encode the enzymes involved in the catabolism of 3-hydroxybenzoate to fumarate and pyruvate through the gentisate pathway. MhbM is a 3-hydroxybenzoate-6-hydroxylase that catalyzed the para-hydroxylation of 3-hydroxybenzoate(3-HBA) to 2,5-dihydroxybenzoate (gentisate). MhbD is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate. MhbI is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. MhbH is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. MhbM is also purified and its dynamics characterization was performed. Frame-shift mutants of mhbR and mhbT were also made to investigate their possible roles in 3-hydroxybenzoate catabolism.
This project is funded by the National Natural Science Foundation of China (No. 30170036). The current result reported here is the second example of the research on
the genes encoding enzymes involved in the entire gentisate pathway and the 3-hydroxybenzoate-6-hydroxylase was studied at molecular level for the first time.
引文
[1] 王修垣译. 生物科学动态，1961.5:25.
[2] http://www.sxdaily.com.cn 陕西日报. 2001.3.24
[3] 林稚兰，黄秀梨. 现代微生物学与实验技术，科学出版社，北京，2000.202-230 .
[4] Tabak H H et al. J Water Pollut Control Fed,1981.53:1503.
[5] Worsey M K et al. J Bacterial, 1975. 124:7.
[6] Chakrabarty A M et al. Science, 1981.214:1133.
[7] Chakrabarty A M et al. Appl Environ Microb, 1982. 44(3):619.
[8] Chakrabarty A M et al. Plasmid in Bacteria. 1984.1.
[9] Chakrabarty A M et al. Genetic Control of Environmental Pollutants. 1984.2.
[10] Chakrabarty A M et al. Genetic Manipulation Impact on Man and Society. 1984:43.
[11] Wong C L et al. Genet Res, 1974. 23:227.
[12] Williams P A et al. J Bacteriol, 1074.120:416.
[13] Fuenmayor, S. L., M. Wild, A. L. Boyes, and P. A. Williams. J. Bacteriol.，1998. 180:2522-2530.
[14] Grund, E., B. Denecke, and R. Eichenlaub. Microbiol. 1992. 58:1874-1877.
[15] Monticello, D. J., D. Bakker, M. Schell, and W. R. Finnerty. Appl. Environ. Microbiol. 1985. 49:761-764.
[16] Cane, P. A., and P. A. Williams. J. Gen. Microbiol. 1986.132:2919-2929.
[17] Grimm, A. C., and C. S. Harwood. J. Bacteriol. 1999. 181:3310-3316.
[18] Hofer, B., S. Backhaus, and K. N. Timmis. Gene 1994. 144:9-16.
[19] Kimura, N., A. Nishi, M. Goto, and K. Furukawa. J. Bacteriol. 1997.179:3936-3943.
[20] Sala-Trepat J M, Evans W C. Eur J Biochem, 1971, 20:400-413.
[21] Stanier R Y, Ornston L N. Adv Microb Physiol, 1973, 9:89-151.
[22] Lack L. Biochimica et Biophysica acta, 1959, 34:117-123.
[23] Tanaka H S, Sugiyama K, Yano K, et al. Agr Chem Soc Jpn Bull, 1957, 21:67-68.
[24] Lack L. J Biol Chem, 1961, 236(11):2835-2840.
[25] Crawford RL, Hutton SW, Chapman PJ. J Bacteriol, 1975, 121(3):794-9.
[26] Bayly R, Chapman P, Dagley S, et al. J Bacteriol, 1980, 143(1):70-77.
[27] Hopper D J, Chapman P J, Dagley S. J Biochem, 1968, 110:79.
[28] Poh C L, Bayly R. J Bacteriol, 1980,143:59-69.
[29] Crawford R L, Frick T D. Appl Environ Microbiol, 1977, 34(2):170-174.
[30] Robson N D, Parrott S, Cooper R A. Microbiology, 1996, 142:2115-2120.
[31] Hagedorn S R, Bradley G, Chapman P J. J Bacteriol, 1985, 163(2):640-647.
[32] Zhou N Y, Fuenmayor S L, Williams P A . J Bacteriol, 2001, 183(2):700-708.
[33] Zhou N Y, Al-Dulayymi J, Baird M S, et al. J Bacteriol, 2002. 184(6):1547-55.
[34] Ribbons,D.W.FEBS Lett 8,1970. 101-104.
[35] Wang LH, Hamzah RY, Yu YM, Tu SC. Biochemistry 1987. 24;26(4):1099-104.
[36] Mashetty SB, Manohar S, Karegoudar TB. Indian J Biochem Biophys, 1996. 33(2):145-8.
[37] Muller JA, Schink B. Arch Microbiol, 2000. 173(4):288-95.
[38] Laempe D, Jahn M, Breese K, Schagger H, Fuchs G. J Bacteriol 2001. 183(3):968-79.
[39] Sumathi Suresh S, Dasgupta D. Biotechnol Prog, 2001. 17(6):1026-31.
[40] Yano K, Arima K. J Gen App. Microbiol, 1958.4:241-258.
[41] Wheelis ML,Pallerom NJ,Stanier BY. Arch Microbiol,1967. 59:302-314.
[42] Goetz, F. E., and L. J. Harmuth. FEMS Microbiol. Lett. 1992. 97:45-49.
[43] Dagley S. Essays in biochemistry, 1975. 81-138.
[44] Jones, D. C. N., and R. A. Cooper. 1990. Arch. Microbiol. 154:489-495.
[45] Monica Suarez, Estrella Ferrer, Amando Garrido-Pertierra, Margarita Martin. FEMS Microbiology Letters, 1995. 126:283-290.
[46] Monica Suarez, Estrella Ferrer, Margarita Martin. FEMS Microbiology Letters, 1996. 143:89-95.
[47] Miller, J. H. Cold Spring Harbor, 1972.N.Y.
[48] F.奥斯曼，R.E.金斯顿等，精编分子生物学实验指南，颜子颖，王海林译,科学出版社, 北京，1998.
[49] Hareland WA, Crawford RL, Chapman PJ, Dagley S. J Bacteriol, 1975. 121:272-285.
[50] Wheatcroft, R., and P. A. Williams. J. Gen. Microbiol. 1981. 124:433-437.
[51] J.萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯，金东雁等译,分子克隆试验指南第二版, 科学出版社,北京，1996.
[52] Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). Cold Spring Harbor, 1989.
[53] PCR技术实验指南，科学出版社，北京，1998.
[54] 聚合链式反应，科学出版社，北京，1998.
[55] Hintner JP, Lechner C, Riegert U, Kuhm AE, Storm T, Reemtsma T, Stolz A. J Bacteriol 2001. 183(23): 6936-42.
[56] Premkumar R., Subba Rao, P.V.,Sreeleela, N.S. and Vaidyanathan,C.S. J.Biochem.1969. 47:825-827.
[57] White-Stevens, R.H. and Kamin,H. J.Biol.Chem.1972.247:2458-2370.
[58] Wang, L.H.,Hamzah,R.Y.,Yu,Y. and Tu,S.C. Biochemistry, 1987. 26,1105-1110.
[59] Graseclose, E.E. and Ribbons,D.W. Biochem. Biophys. Res.Commun.1973. 55,897-903.
[60] Rajasekharan, S., Rajasekharan, R. and Vaidyanathan, C.S. Arch. Biochem. Biophys. 1990.278,21-25.
[61] Feng Y, Khoo H E, Poh CL. Appl Environ Microbiol, 1999, 65(3):946-50.
[62] Fu W, Oriel P. Extremophiles, 1998, 2(4):439-46.
[63] Harpel M R, Lipscomb J D. J Biol Chem, 1990, 265(36):22187-96.8-800.
[64] Werwath J, Arfmann H A, Pieper D H, et al. J Bacteriol, 1998, 180(16):4171-6.
[65] Chua C H, Feng Y, Yeo C C, et al. FEMS Microbiol Lett, 2001, 204(1):141-6.
[66] Suarez M, Ferrer E, Martin M. FEMS Microbiol Lett, 1996, 143(1):89-95.
[2] http://www.sxdaily.com.cn 陕西日报. 2001.3.24
[3] 林稚兰，黄秀梨. 现代微生物学与实验技术，科学出版社，北京，2000.202-230 .
[4] Tabak H H et al. J Water Pollut Control Fed,1981.53:1503.
[5] Worsey M K et al. J Bacterial, 1975. 124:7.
[6] Chakrabarty A M et al. Science, 1981.214:1133.
[7] Chakrabarty A M et al. Appl Environ Microb, 1982. 44(3):619.
[8] Chakrabarty A M et al. Plasmid in Bacteria. 1984.1.
[9] Chakrabarty A M et al. Genetic Control of Environmental Pollutants. 1984.2.
[10] Chakrabarty A M et al. Genetic Manipulation Impact on Man and Society. 1984:43.
[11] Wong C L et al. Genet Res, 1974. 23:227.
[12] Williams P A et al. J Bacteriol, 1074.120:416.
[13] Fuenmayor, S. L., M. Wild, A. L. Boyes, and P. A. Williams. J. Bacteriol.，1998. 180:2522-2530.
[14] Grund, E., B. Denecke, and R. Eichenlaub. Microbiol. 1992. 58:1874-1877.
[15] Monticello, D. J., D. Bakker, M. Schell, and W. R. Finnerty. Appl. Environ. Microbiol. 1985. 49:761-764.
[16] Cane, P. A., and P. A. Williams. J. Gen. Microbiol. 1986.132:2919-2929.
[17] Grimm, A. C., and C. S. Harwood. J. Bacteriol. 1999. 181:3310-3316.
[18] Hofer, B., S. Backhaus, and K. N. Timmis. Gene 1994. 144:9-16.
[19] Kimura, N., A. Nishi, M. Goto, and K. Furukawa. J. Bacteriol. 1997.179:3936-3943.
[20] Sala-Trepat J M, Evans W C. Eur J Biochem, 1971, 20:400-413.
[21] Stanier R Y, Ornston L N. Adv Microb Physiol, 1973, 9:89-151.
[22] Lack L. Biochimica et Biophysica acta, 1959, 34:117-123.
[23] Tanaka H S, Sugiyama K, Yano K, et al. Agr Chem Soc Jpn Bull, 1957, 21:67-68.
[24] Lack L. J Biol Chem, 1961, 236(11):2835-2840.
[25] Crawford RL, Hutton SW, Chapman PJ. J Bacteriol, 1975, 121(3):794-9.
[26] Bayly R, Chapman P, Dagley S, et al. J Bacteriol, 1980, 143(1):70-77.
[27] Hopper D J, Chapman P J, Dagley S. J Biochem, 1968, 110:79.
[28] Poh C L, Bayly R. J Bacteriol, 1980,143:59-69.
[29] Crawford R L, Frick T D. Appl Environ Microbiol, 1977, 34(2):170-174.
[30] Robson N D, Parrott S, Cooper R A. Microbiology, 1996, 142:2115-2120.
[31] Hagedorn S R, Bradley G, Chapman P J. J Bacteriol, 1985, 163(2):640-647.
[32] Zhou N Y, Fuenmayor S L, Williams P A . J Bacteriol, 2001, 183(2):700-708.
[33] Zhou N Y, Al-Dulayymi J, Baird M S, et al. J Bacteriol, 2002. 184(6):1547-55.
[34] Ribbons,D.W.FEBS Lett 8,1970. 101-104.
[35] Wang LH, Hamzah RY, Yu YM, Tu SC. Biochemistry 1987. 24;26(4):1099-104.
[36] Mashetty SB, Manohar S, Karegoudar TB. Indian J Biochem Biophys, 1996. 33(2):145-8.
[37] Muller JA, Schink B. Arch Microbiol, 2000. 173(4):288-95.
[38] Laempe D, Jahn M, Breese K, Schagger H, Fuchs G. J Bacteriol 2001. 183(3):968-79.
[39] Sumathi Suresh S, Dasgupta D. Biotechnol Prog, 2001. 17(6):1026-31.
[40] Yano K, Arima K. J Gen App. Microbiol, 1958.4:241-258.
[41] Wheelis ML,Pallerom NJ,Stanier BY. Arch Microbiol,1967. 59:302-314.
[42] Goetz, F. E., and L. J. Harmuth. FEMS Microbiol. Lett. 1992. 97:45-49.
[43] Dagley S. Essays in biochemistry, 1975. 81-138.
[44] Jones, D. C. N., and R. A. Cooper. 1990. Arch. Microbiol. 154:489-495.
[45] Monica Suarez, Estrella Ferrer, Amando Garrido-Pertierra, Margarita Martin. FEMS Microbiology Letters, 1995. 126:283-290.
[46] Monica Suarez, Estrella Ferrer, Margarita Martin. FEMS Microbiology Letters, 1996. 143:89-95.
[47] Miller, J. H. Cold Spring Harbor, 1972.N.Y.
[48] F.奥斯曼，R.E.金斯顿等，精编分子生物学实验指南，颜子颖，王海林译,科学出版社, 北京，1998.
[49] Hareland WA, Crawford RL, Chapman PJ, Dagley S. J Bacteriol, 1975. 121:272-285.
[50] Wheatcroft, R., and P. A. Williams. J. Gen. Microbiol. 1981. 124:433-437.
[51] J.萨姆布鲁克，E.F.弗里奇，T.曼尼阿蒂斯，金东雁等译,分子克隆试验指南第二版, 科学出版社,北京，1996.
[52] Sambrook, J., E. F. Fritsch, and T. Maniatis (ed.). Cold Spring Harbor, 1989.
[53] PCR技术实验指南，科学出版社，北京，1998.
[54] 聚合链式反应，科学出版社，北京，1998.
[55] Hintner JP, Lechner C, Riegert U, Kuhm AE, Storm T, Reemtsma T, Stolz A. J Bacteriol 2001. 183(23): 6936-42.
[56] Premkumar R., Subba Rao, P.V.,Sreeleela, N.S. and Vaidyanathan,C.S. J.Biochem.1969. 47:825-827.
[57] White-Stevens, R.H. and Kamin,H. J.Biol.Chem.1972.247:2458-2370.
[58] Wang, L.H.,Hamzah,R.Y.,Yu,Y. and Tu,S.C. Biochemistry, 1987. 26,1105-1110.
[59] Graseclose, E.E. and Ribbons,D.W. Biochem. Biophys. Res.Commun.1973. 55,897-903.
[60] Rajasekharan, S., Rajasekharan, R. and Vaidyanathan, C.S. Arch. Biochem. Biophys. 1990.278,21-25.
[61] Feng Y, Khoo H E, Poh CL. Appl Environ Microbiol, 1999, 65(3):946-50.
[62] Fu W, Oriel P. Extremophiles, 1998, 2(4):439-46.
[63] Harpel M R, Lipscomb J D. J Biol Chem, 1990, 265(36):22187-96.8-800.
[64] Werwath J, Arfmann H A, Pieper D H, et al. J Bacteriol, 1998, 180(16):4171-6.
[65] Chua C H, Feng Y, Yeo C C, et al. FEMS Microbiol Lett, 2001, 204(1):141-6.
[66] Suarez M, Ferrer E, Martin M. FEMS Microbiol Lett, 1996, 143(1):89-95.