猪链球菌3-羟基-3-甲基戊二酰辅酶A还原酶的原核表达、活性检测和同源建模

doi:10.11843/j.issn.0366-6964.2016.11.021

摘要/Abstract

摘要：

旨在以猪链球菌（Streptococcus suis, S. suis）3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）为药靶，给S. suis病防治研制新型抗菌药提供理论和试验基础。原核表达了S. suis HMGR，用Ni-NTA树脂对重组蛋白质进行了纯化，并用分光光度法测定了其酶活力。此外，运用CLUSTALW对同源HMGR氨基酸序列进行多序列比对。通过SWISS-MODEL对S. suis HMGR三维结构进行同源建模，并用PROCHECK评价模型的质量。酶活力测定结果显示，催化HMG-CoA还原为甲羟戊酸的反应中，S. suis HMGR最佳反应温度和pH分别为37 ℃和5.0，其Vmax和Km为846 U•mg^-1和0.213 mmol•L^－1。经分析显示，肺炎链球菌HMGR晶体结构（PDB ID：3QAE_A）是构建S. suis HMGR三维结构的最佳模板。构建的三维结构模型经质量评估证实为可靠的理论模型，将有助于虚拟筛选出对抗S. suis感染的新型抗生素，酶活力的测定为用试验手段验证S. suis HMGR抑制剂的有效性提供了可行性的方法。

Abstract:

The aim of this study was to take the Streptococcus suis (S. suis) 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) as a drug target, and provide theoretical and experimental basis for the development of novel antibiotics for the prevention and treatment of S. suis infection. In this experiment, S. suis HMGR was prokaryotically expressed and purified by Ni-NTA agrose. Its enzyme activity was determined by spectrophotometric method. In addition, a multiple sequence alignment of HMGR was performed by CLUSTALW to identify conserved domains. Homology modeling of the three-dimensional (3D) structure of S. suis HMGR was performed by SWISS-MODEL. The quality of the model was evaluated by PROCHECK. The result of enzyme activity analyses showed that optimal reaction conditions of S. suis HMGR was pH 5.0 and 37 ℃, the Vmax and Km value was 846 U•mg^-1 and 0.213 mmol•L^－1 respectively. The analysis of SWISS-MODEL showed that crystal structure of Streptococcus pneumonia HMGR was the appropriate template for homology modeling of S. suis HMGR. The theoretic model of 3D structure of S. suis HMGR was proven to be reliable through quality assessment. The S. suis HMGR model will contribute to virtual screening of novel antibacterial agents against S. suis infection. Enzyme activity assay provides a feasible method for validation of effective inhibitors against S. suis HMGR in biochemical experiment.

中图分类号:

S852.61

刘璨颖，徐卓菲，付强. 猪链球菌3-羟基-3-甲基戊二酰辅酶A还原酶的原核表达、活性检测和同源建模[J]. 畜牧兽医学报, 2016, 47(11): 2318-2324.

LIU Can-ying, XU Zhuo-fei, FU Qiang. Prokaryotic Expression, Activity Assay and Homology Modeling of the 3-hydroxy-3-methylglutaryl Coenzyme A Reductase from Streptococcus suis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(11): 2318-2324.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2016.11.021

https://www.xmsyxb.com/CN/Y2016/V47/I11/2318

参考文献

［1］WINDSOR R S, ELLIOTT S D. Streptococcal infection in young pigs. IV. An outbreak of streptococcal meningitis in weaned pigs［J］. J Hyg (Lond), 1975, 75(1): 69-78.
［2］WINDSOR R S. Meningitis in pigs caused by Streptococcus suis type 2［J］. Vet Rec, 1977, 101（19）: 378-379.
［3］ERICKSON E D. Streptococcosis［J］. J Am Vet Med Assoc, 1987, 191（11）: 1391-1393.
［4］HIGGINS R, GOTTSCHALK M, MITTAL K R, et al. Streptococcus suis infection in swine. A sixteen month study［J］. Can J Vet Res, 1990, 54（1）: 170-173.
［5］CHANTER N, JONES P W, ALEXANDER T J. Meningitis in pigs caused by Streptococcus suis-a speculative review［J］. Vet Microbiol, 1993, 36（1-2）: 39-55.
［6］GOYETTE-DESJARDINS G, AUGER J P, XU J, et al. Streptococcus suis, an important pig pathogen and emerging zoonotic agent-an update on the worldwide distribution based on serotyping and sequence typing［J］. Emerg Microbes Infect, 2014, 3 (6): e45.
［7］WERTHEIM H F, NGUYEN H N, TAYLOR W, et al. Streptococcus suis, an important cause of adult bacterial meningitis in northern Vietnam［J］. PLoS One, 2009, 4(6):e5973.
［8］REAMS R Y, GLICKMAN L T, HARRINGTON D D, et al. Streptococcus suis infection in swine: a retrospective study of 256 cases. Part I. Epidemiologic factors and antibiotic susceptibility patterns［J］. J Vet Diag Invest, 1993, 5（3）: 363-367.
［9］HEUSTON S, BEGLEY M, GAHAN C G, et al. Isoprenoid biosynthesis in bacterial pathogens［J］. Microbiology, 2012, 158(Pt6):1389-1401.
［10］REUSCH V M Jr. Lipopolymers, isoprenoids, and the assembly of the gram-positive cell wall［J］. Crit Rev Microbiol, 1984, 11（2）:129-155.
［11］MATSUMI R, ATOMI H, DRIESSEN A J, et al. Isoprenoid biosynthesis in Archaea-biochemical and evolutionary implications［J］. Res Microbiol, 2011, 162（1）: 39-52.
［12］BOUCHER Y, DOOLITTLE W F. The role of lateral gene transfer in the evolution of isoprenoid biosynthesis pathways［J］. Mol Microbiol, 2000, 37(4): 703-716.
［13］SETO H, WATANABE H, FURIHATA K. Simultaneous operation of the mevalonate and non-mevalonate pathways in the biosynthesis of isopentenyl diphosphate in Streptomyces aeriouvifer［J］. Tetrahedron Lett, 1996, 37（44）: 7979-7982.
［14］WILDING E I, BROWN J R, BRYANT A P, et al. Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci［J］. J Bacteriol, 2000, 182（15）:4319-4327.
［15］WILDING E I, KIM D Y, BRYANT A P, et al. Essentiality, expression, and characterization of the Class II 3-hydroxy-3-methylglutaryl coenzymeA reductase of Staphylococcus aureus［J］. J Bacteriol, 2000, 182(18): 5147-5152.
［16］BOCHAR D A, STAUFFACHER C V, RODWELL V W. Sequence comparisons reveal two classes of 3-hydroxy-3-methylglutaryl coenzymeA reductase［J］. Mol Genet Metab, 1999, 66(2):122-127.
［17］MIZIORKO H M. Enzymes of the mevalonate pathway of isoprenoid biosynthesis［J］. Arch Biochem Biophys, 2011, 505(2): 131-143.
［18］LAWRENCE C M, RODWELL V W, STAUFFACHER C V. Crystalstructure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution［J］. Science, 1995, 268(5218):1758-1762.
［19］ISTVAN E S. Bacterial and mammalian HMG-CoA reductases: related enzymes with distinct architectures［J］. Curr Opin Struct Biol, 2001, 11(6): 746-751.
［20］ALBERTS A W, CHEN J, KURON G, et al. Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent［J］. Proc Natl Acad Sci U S A, 1980, 77(7): 3957-3961.
［21］LI W, HU X Y, LIU L, et al. Induction of protective immune response against Streptococcus suis serotype 2 infection by the surface antigen HP0245［J］. FEMS Microbiol Lett, 2011, 316(2): 115-122.
［22］李鹏, 陈兰英. 血脂康抑制猪肝HMG-CoA还原酶的活力［J］. 基础医学与临床, 2003, 23(5): 531-534.
LI P, CHEN L Y. Xuezhikang inhibits the activity of HMG-CoA reductase in pig liver［J］. Basic Medical Sciences and Clinics, 2003, 23(5): 531-534.（in Chinese）
［23］LI K B. ClustalW-MPI: ClustalW analysis using distributed and parallel computing［J］. Bioinformatics, 2003, 19(12): 1585-1586.
［24］BIASINI M, BIENERT S, WATERHOUSE A, et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information［J］. Nucleic Acids Res, 2014, 42: 252-258.
［25］LASKOWSKI R A, MACARTHUR M W, MOSS D S, et al. PROCHECK: a program to check the stereochemical quality of protein structures［J］. J Appl Cryst, 1993, 26(2): 283-291.
［26］FENG Y J, ZHANG H M, WU Z W, et al. Streptococcus suis infection an emerging/reemerging challenge of bacterial infectious diseases?［J］. Virulence, 2014, 5(4): 477-497
［27］LI D, GUI J, LI Y J, et al. Structure-based design and screen of novel inhibitors for class II 3-hydroxy-3-methylglutaryl coenzyme A reductase from Streptococcus pneumoniae［J］. J Chem Inf Model, 2012, 52(7): 1833-1841.
［28］BEN Y L, CUI G Z, LI C, et al. Expression, purification, characteristics and homology modeling of the HMGS from Streptococcus penumoniae［J］. Biomed Environ Sci, 2009, 22(3): 229-236.

[1]	吕若一, 司晓慧, 孙志刚, 史晓敏, 刘晓晔. 猪链球菌耐药现状分析及感染防控措施[J]. 畜牧兽医学报, 2023, 54(12): 4920-4933.
[2]	支岩, 梅晨, 刘珍邑, 乌云格日乐, 王宏俊, 胡格. 副鸡禽杆菌毒力因子研究进展[J]. 畜牧兽医学报, 2023, 54(12): 4934-4942.
[3]	王佳宁, 张自强, 孔德婧, 冯彩彩, 张飞可, 刘玉梅. 家兔肺炎克雷伯菌的分离鉴定[J]. 畜牧兽医学报, 2023, 54(12): 5198-5206.
[4]	李莉莉, 陈凯风, 陈兵, 周洲平, 王南威, 瞿孝云, 徐成刚, 廖明, 张建民. STM1827在鼠伤寒沙门菌生物被膜形成及环境应激中的调控作用[J]. 畜牧兽医学报, 2023, 54(12): 5207-5217.
[5]	杨梦林, 郑世奇, 彭凯, 王玮, 黄燕华, 彭杰. 鸽源鼠伤寒沙门菌的分离鉴定及致病性分析[J]. 畜牧兽医学报, 2023, 54(11): 4880-4888.
[6]	武周慧, 王瑜, 杜衡, 王之文, 肖爽, 武金亮, 王真. 替拉扎明对多重耐药沙门菌抗菌增敏活性分析[J]. 畜牧兽医学报, 2023, 54(10): 4362-4371.
[7]	刘鑫欢, 恽佳蕾, 毛立, 李基棕, 郝飞, 何苗锋, 杨蕾蕾, 张纹纹, 程子龙, 孙敏, 刘茂军, 王少辉, 白娟, 李文良. 羊腹泻样本中大肠杆菌的分离、毒力基因与耐药性分析[J]. 畜牧兽医学报, 2023, 54(8): 3445-3454.
[8]	胡秀花, 孙芷馨, 赵梦洋, 谢佳琪, 王敏, 陈海良, 葛昕, 刘天龙, 王少林. 野生松鼠源屎肠球菌的致病性与耐药性分析[J]. 畜牧兽医学报, 2023, 54(7): 3012-3021.
[9]	赵菲菲, 李杰, 韩宁, 谢仕廷, 曾振灵. 分离自屠宰场的肺炎克雷伯菌的耐药性分析[J]. 畜牧兽医学报, 2023, 54(7): 3044-3053.
[10]	陈松彪, 尚珂, 杜付熙, 余祖华, 李静, 贾艳艳, 廖成水, 张春杰, 丁轲, 程相朝. 沙门菌VI型分泌系统组装、结构特征和分泌调控网络的研究进展[J]. 畜牧兽医学报, 2023, 54(6): 2252-2263.
[11]	曾成容, 王娜, 毕文文, 梅世慧, 何广霞, 张峻杰, 陈泽, 文明, 周碧君. A型产气荚膜梭菌感染鸭回肠代谢组学分析[J]. 畜牧兽医学报, 2023, 54(6): 2555-2569.
[12]	李蔚, 张强, 瞿嘉豪, 吴亚平, 胡若辰, 贾若艺, 郭如海, 马清义, 潘广林, 王兴龙. 大熊猫肠道菌群年龄演替规律分析[J]. 畜牧兽医学报, 2023, 54(6): 2619-2630.
[13]	翟云逸, 袁野, 李俊玫, 田路路, 刁梓洋, 李彬, 陈家露, 周栋, 靳亚平, 王爱华. 布鲁氏菌外膜蛋白16单克隆抗体的制备及初步应用[J]. 畜牧兽医学报, 2023, 54(5): 2083-2091.
[14]	田艳红, 于江旭, 焦宇洲, 高东阳, 蔡旭旺. 沙门菌脂多糖的结构修饰及其效应的研究进展[J]. 畜牧兽医学报, 2023, 54(4): 1392-1402.
[15]	陈烨馨, 谢梦圆, 李文豪, 张志丹, 王晓丹, 陈柯佳, 刘平平, 周伟光, 王建龙, 徐晓静. 牛轮状病毒和志贺菌阳性犊牛腹泻粪便样本中肠道菌群的分析[J]. 畜牧兽医学报, 2023, 54(4): 1624-1631.