Ensifer meliloti 1021烟酰胺酶的酶学特性及3-氰基吡啶调控机理的研究
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摘要
克隆和异源表达固氮菌Ensifer meliloti 1021烟酰胺酶(NAMase),研究其酶学特性,并探究3-氰基吡啶(3-CP)对该酶的调控机制。PCR扩增获得E. meliloti 1021烟酰胺酶基因(nam)序列,构建含nam的重组质粒并导入Escherichia coli Rosetta(DE3)中异源表达,用Ni-NTA亲和层析纯化蛋白,探究温度、pH、有机溶剂及金属离子对NAMase活性的影响,并对烟酰胺酶进行了固定化研究。结果显示,克隆得到长636 bp的nam,编码的蛋白分子量为22.6 kD,等电点(PI)为5.5。NAMase的最适pH为7.0,在30-50℃温度范围内孵育2 h酶活维持在97.1%以上,最适温度在30-70℃。Ag2+和异戊醇对NAMase的活性抑制率最大。在E. meliloti 1021野生菌株及NAMase过表达菌株转化烟酰胺的过程中3-CP会抑制烟酸的产量,这种效应并非底物抑制作用。
This work aims to clone and express the NAMase of Ensifer meliloti 1021,and to investigate its enzymatic properties and explore the enzymatic mechanisms regulated by 3-CP. PCR was used to amplify the gene nam of E. meliloti 1021 and the constructed recombinant nam-containing plasmid was imported to Escherichia coli Rosetta(DE3)cells for heterologous expression. Ni-NTA affinity chromatography was employed to purify the protein. The effects of temperature,pH,organic solvents and metal ions on NAMase activity were explored,and the immobilization of NAMase was further investigated. The results showed that the full-length of nam was 636 bp,encoded a22.6 kD protein with PI of 5.5. The optimal pH for NAMase was 7,it retained over 97.1% of its initial activity after incubation at 30-50℃ for 2 h,and the optimal temperature ranged in 30-70℃. Ag2+ and isoamyl alcohol demonstrated the highest inhibitory effect on NAMase activity. 3-CP inhibited nicotinic acid production during the transformation of nicotinamide by E. meliloti 1021 and over-expressed NAMase in E. coli Rosetta(DE3),and this was not a substrate inhibition.
This work aims to clone and express the NAMase of Ensifer meliloti 1021,and to investigate its enzymatic properties and explore the enzymatic mechanisms regulated by 3-CP. PCR was used to amplify the gene nam of E. meliloti 1021 and the constructed recombinant nam-containing plasmid was imported to Escherichia coli Rosetta(DE3)cells for heterologous expression. Ni-NTA affinity chromatography was employed to purify the protein. The effects of temperature,pH,organic solvents and metal ions on NAMase activity were explored,and the immobilization of NAMase was further investigated. The results showed that the full-length of nam was 636 bp,encoded a22.6 kD protein with PI of 5.5. The optimal pH for NAMase was 7,it retained over 97.1% of its initial activity after incubation at 30-50℃ for 2 h,and the optimal temperature ranged in 30-70℃. Ag2+ and isoamyl alcohol demonstrated the highest inhibitory effect on NAMase activity. 3-CP inhibited nicotinic acid production during the transformation of nicotinamide by E. meliloti 1021 and over-expressed NAMase in E. coli Rosetta(DE3),and this was not a substrate inhibition.
引文
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[10]Belenky P, Bogan KL, Brenner C. NAD+metabolism in health and disease[J]. Trends in Biochemical Sciences, 2007, 32(1):12-19.
[11]Smith BC, Anderson MA, Hoadley KA, et al. Structural and kinetic isotope effect studies of nicotinamidase(Pnc1)from Saccharomyces cerevisiae[J]. Biochemistry, 2012, 51(1):243-256.
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[13]Rogina B, Helfand SL, Ames BN. Sir2 mediates longevity in the fly through a pathway related to calorie restriction[J]. Proc Natl Acad Sci USA, 2004, 101(45):15998-16003.
[14]Ge F, Zhou LY, Wang Y, et al. Hydrolysis of the neonicotinoid insecticide thiacloprid by the N2-fixing bacterium Ensifer meliloti CGMCC 7333[J]. International Biodeterioration&Biodegradation, 2014, 93:10-17.
[15]Mathew CD, Nagasawa T, Kobayashi M, et al. Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1[J]. Applied&Environmental Microbiology,1988, 54(4):1030-1032.
[16]Sánchezcarrón G, Garcíagarcía MI, Zapatapérez R, et al.Biochemical and mutational analysis of a novel nicotinamidase from Oceanobacillus iheyensis HTE831[J]. PLoS One, 2013, 8(2):e56727.
[17]Pardee AB, Benz EJ, Stpeter DA, et al. Hyperproduction and purification of nicotinamide deamidase, a microconstitutive enzyme of Escherichia coli[J]. Journal of Biological Chemistry, 1971,246(22):6792-6796.
[18]Yan C, Sloan DL. Purification and characterization of nicotinamide deamidase from yeast[J]. Journal of Biological Chemistry, 1987,262(19):9082-9087.
[19]Somoskovi A, Wade MM, Sun Z, et al. Iron enhances the antituberculous activity of pyrazinamide[J]. Journal of Antimicrobial Chemotherapy, 2004, 53(2):192-196.
[20]Zhang H, Deng JY, Bi LJ, et al. Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase[J]. FEBS Journal,2008, 275(4):753-762.
[21]Ion BF, Kazim E, Gauld JW. A multi-scale computational study on the mechanism of streptococcus pneumoniae nicotinamidase(SpNic)[J]. Molecules, 2014, 19(10):15735-15752.
[22]Frothingham R, Meekero’Connell WA, Talbot EA, et al.Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA[J].Antimicrobial Agents&Chemotherapy, 1996, 40(6):1426-1431.
[23]Sheen P, Ferrer P, Gilman RH, et al. Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase[J].American Journal of Tropical Medicine&Hygiene, 2012, 87(1):153-161.
[24]Ahmady A, Poolad T, Rafee P, et al. Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis[J]. Tuberk Toraks, 2013,61(2):110-114.
[2]Kim SH, Oriel P. Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli[J].Enzyme and Microbial Technology, 2000, 27:492-501.
[3]Alfani F, Cantarella M, Spera A. et al. Operational stability of Brevibacterium imperalis CBS489-74 nitrile hydratase[J]. Journal of Molecular Catalysis(B:Enzymatic), 2001, 11(4/6):687-697.
[4]Park HJ, Park H, Rutger JF, et al. Characterization of nitrile hydrolyzing enzymes produced from Rhodococcus erythropolis[J].Korean Society for Microbiology and Biotechnology, 2006, 34(3):204-210.
[5]Prasad S, Bhalla TC. Nitrile hydratases(NHases):At the interface of academia and industry[J]. Biotechnology Advances, 2010, 28(6):725-741.
[6]Pace HC, Brenner C. The nitrilase superfamily:classification,structure and function. Genome Biology, 2001, 2(1):1-9.
[7]Anderson RM, Bitterman KJ, Wood JG, et al. Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae[J]. Nature, 2003, 423(6936):181-185.
[8]French JB, Cen Y, Sauve AA, et al. High-resolution crystal structures of Streptococcus pneumoniae nicotinamidase with trapped intermediates provide insights into the catalytic mechanism and inhibition by aldehydes[J]. Biochemistry, 2010, 49(40):8803-8812.
[9]Rongvaux A, Shea RJ, Mulks MH, et al. Pre-B-cell colony-enhancing factor, whose expression is up-regulated in activated lymphocytes,is a nicotinamide phosphoribosyl transferase, a cytosolic enzyme involved in NAD biosynthesis[J]. European Of Immunology,2002, 11(32), 3225-3234.
[10]Belenky P, Bogan KL, Brenner C. NAD+metabolism in health and disease[J]. Trends in Biochemical Sciences, 2007, 32(1):12-19.
[11]Smith BC, Anderson MA, Hoadley KA, et al. Structural and kinetic isotope effect studies of nicotinamidase(Pnc1)from Saccharomyces cerevisiae[J]. Biochemistry, 2012, 51(1):243-256.
[12]Balan V, Miller GS, Kaplun L, et al. Life span extension and neuronal cell protection by drosophila nicotinamidase[J].Journal of Biological Chemistry, 2008, 283(41):27810-27819.
[13]Rogina B, Helfand SL, Ames BN. Sir2 mediates longevity in the fly through a pathway related to calorie restriction[J]. Proc Natl Acad Sci USA, 2004, 101(45):15998-16003.
[14]Ge F, Zhou LY, Wang Y, et al. Hydrolysis of the neonicotinoid insecticide thiacloprid by the N2-fixing bacterium Ensifer meliloti CGMCC 7333[J]. International Biodeterioration&Biodegradation, 2014, 93:10-17.
[15]Mathew CD, Nagasawa T, Kobayashi M, et al. Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1[J]. Applied&Environmental Microbiology,1988, 54(4):1030-1032.
[16]Sánchezcarrón G, Garcíagarcía MI, Zapatapérez R, et al.Biochemical and mutational analysis of a novel nicotinamidase from Oceanobacillus iheyensis HTE831[J]. PLoS One, 2013, 8(2):e56727.
[17]Pardee AB, Benz EJ, Stpeter DA, et al. Hyperproduction and purification of nicotinamide deamidase, a microconstitutive enzyme of Escherichia coli[J]. Journal of Biological Chemistry, 1971,246(22):6792-6796.
[18]Yan C, Sloan DL. Purification and characterization of nicotinamide deamidase from yeast[J]. Journal of Biological Chemistry, 1987,262(19):9082-9087.
[19]Somoskovi A, Wade MM, Sun Z, et al. Iron enhances the antituberculous activity of pyrazinamide[J]. Journal of Antimicrobial Chemotherapy, 2004, 53(2):192-196.
[20]Zhang H, Deng JY, Bi LJ, et al. Characterization of Mycobacterium tuberculosis nicotinamidase/pyrazinamidase[J]. FEBS Journal,2008, 275(4):753-762.
[21]Ion BF, Kazim E, Gauld JW. A multi-scale computational study on the mechanism of streptococcus pneumoniae nicotinamidase(SpNic)[J]. Molecules, 2014, 19(10):15735-15752.
[22]Frothingham R, Meekero’Connell WA, Talbot EA, et al.Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA[J].Antimicrobial Agents&Chemotherapy, 1996, 40(6):1426-1431.
[23]Sheen P, Ferrer P, Gilman RH, et al. Role of metal ions on the activity of Mycobacterium tuberculosis pyrazinamidase[J].American Journal of Tropical Medicine&Hygiene, 2012, 87(1):153-161.
[24]Ahmady A, Poolad T, Rafee P, et al. Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis[J]. Tuberk Toraks, 2013,61(2):110-114.
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