[1]荆雅玮,左佳坤,王志豪,等.氧化型辅酶NAD+及布氏杆菌SahH活性位点对SahH催化活性的影响[J].南京农业大学学报,2020,43(5):903-909.[doi:10.7685/jnau.201910013]
 JING Yawei,ZUO Jiakun,WANG Zhihao,et al.Effects of oxidized coenzyme NAD+ and SahH active site on catalytic activity of Brucella SahH[J].Journal of Nanjing Agricultural University,2020,43(5):903-909.[doi:10.7685/jnau.201910013]
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氧化型辅酶NAD+及布氏杆菌SahH活性位点对SahH催化活性的影响()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
43卷
期数:
2020年5期
页码:
903-909
栏目:
动物科学
出版日期:
2020-09-15

文章信息/Info

Title:
Effects of oxidized coenzyme NAD+ and SahH active site on catalytic activity of Brucella SahH
作者:
荆雅玮1 左佳坤12 王志豪1 胡剑刚1 黄燕1 米荣升1 苗晋锋2 PHOUTHAPANE Vanhnaseng3 陈兆国1 韩先干1
1. 中国农业科学院上海兽医研究所, 上海 200241;
2. 南京农业大学动物医学院, 江苏 南京 210095;
3. 老挝科技部生态与生物技术研究所, 万象 22797
Author(s):
JING Yawei1 ZUO Jiakun12 WANG Zhihao1 HU Jiangang1 HUANG Yan1 MI Rongsheng1 MIAO Jinfeng2 PHOUTHAPANE Vanhnaseng3 CHEN Zhaoguo1 HAN Xiangan1<
1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;
3. Animal Science Center at Biotechnology and Ecology Institute, Ministry of Science and Technology of Laos, Vientiane 22797, Laos
关键词:
布氏杆菌S-腺苷同型半胱氨酸水解酶酶活性活性位点
Keywords:
BrucellaS-adenosylhomocysteine hydrolase(SahH)enzyme activityactive site
分类号:
S855.1+2
DOI:
10.7685/jnau.201910013
摘要:
[目的] 本文旨在鉴定影响布氏杆菌S-腺苷同型半胱氨酸水解酶(S-adenosylhomocysteine hydrolase,SahH)催化S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)产生同型半胱氨酸(homocysteine,HCY)的催化活性位点。[方法] 将表达载体pET28a-Bru-sahH转化大肠杆菌BL21中,表达、纯化布氏杆菌SahH(Bru-SahH)蛋白,分析辅酶NAD+、磷酸化修饰及活性位点对Bru-SahH催化活性的影响。[结果] 添加NAD+后,Bru-SahH的催化活性降低85.6%;对Bru-SahH质谱分析表明,其444位丝氨酸为可能的磷酸化位点,对该位点突变后Bru-SahH催化SAH形成HCY的活性降低85.5%;生物信息学分析表明,Bru-SahH的337位和338位氨基酸为其活性位点,分别对上述2个位点进行单突变和双突变,突变后的Bru-SahH催化活性降低90.8%以上。[结论] Bru-SahH的337、338和444位氨基酸是其催化活性位点,添加外源性NAD+可抑制其催化活性。
Abstract:
[Objectives] The objective of this paper is to investigate the influencing factors of catalytic activity and identification of active catalytic site of Brucella S-adenosylhomocysteine hydrolase (SahH).[Methods] The expression vector pET28a-Bru-sahH was transformed into Escherich coli BL21,the influencing factors of catalytic activity of SahH in vitro were analyzed by addition of exogenous coenzyme NAD+,analysis of modification and identification of catalytic activity sites of SahH,respectively.[Results] The results showed that the catalytic activity of recombinant Brucella protein SahH (Bru-SahH) was decreased by 85.6% by addition of exogenous NAD+. Furthermore,the catalytic activity of Bru-SahH was decreased by 85.5% by mutation of 444th amino acid,which was a possible phosphorylation modification by mass spectrum analysis of Bru-SahH. In addition,the catalytic activity of Bru-SahH was decreased more than 90.8% by single or double site mutation at 337th and/or 338th amino acids of Bru-SahH,which was predicted to the key activity sites of Bru-SahH by bioinformatics analysis.[Conclusions] The results indicated that the key catalytic sites of Bru-SahH were 337th,338th and 444th amino acids. Exogenous NAD+ could inhibit catalytic activity of Bru-SahH.

参考文献/References:

[1] Atluri V L,Xavier M N,de Jong M F,et al. Interactions of the human pathogenic Brucella species with their hosts[J]. Annual Review of Microbiology,2011,65:523-541.
[2] Shao H,Lamont R J,Demuth D R. Autoinducer 2 is required for biofilm growth of Aggregatibacter(Actinobacillus)actinomycetemcomitans[J]. Infection and Immunity,2007,75(9):4211-4218.
[3] Walters M,Sircili M P,Sperandio V. AI-3 synthesis is not dependent on luxS in Escherichia coli[J]. Journal of Bacteriology,2006,188(16):5668-5681.
[4] Leal J F,Ferrer I,Blanco-Aparicio C,et al. S-adenosylhomocysteine hydrolase downregulation contributes to tumorigenesis[J]. Carcinogenesis,2008,29(11):2089-2095.
[5] Baric I. Inherited disorders in the conversion of methionine to homocysteine[J]. Journal of Inherited Metabolic Disease,2009,32(4):459-471.
[6] de Clercq E. John Montgomery’s legacy:carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral activity[J]. Nucleosides,Nucleotides & Nucleic Acids,2005,24(10/11/12):1395-1415.
[7] Redanz S,Standar K,Podbielski A,et al. Heterologous expression of sahH reveals that biofilm formation is autoinducer-2-independent in Streptococcus sanguinis but is associated with an intact activated methionine cycle[J]. The Journal of Biological Chemistry,2012,287(43):36111-36122.
[8] Han T,Li Y,Shan Q,et al. Characterization of S-adenosylhomocysteine/methylthioadenosine nucleosidase on secretion of AI-2 and biofilm formation of Escherichia coli[J]. Microbial Pathogenesis,2017,108:78-84.
[9] Manszewski T,Singh K,Imiolczyk B,et al. An enzyme captured in two conformational states:crystal structure of S-adenosyl-L-homocysteine hydrolase from Bradyrhizobium elkanii[J]. Acta crystallographica section D:Biological Crystallography,2015,71:2422-2432.
[10] Brzezinski K,Czyrko J,Sliwiak J,et al. S-adenosyl-L-homocysteine hydrolase from a hyperthermophile(Thermotoga maritima)is expressed in Escherichia coli in inactive form:Biochemical and structural studies[J]. International Journal of Biological Macromolecules,2017,104:584-596.
[11] 徐达,吴小卡,荆雅玮,等. 布氏杆菌半胱氨酸水解酶在甲硫氨酸循环中的催化活性研究[J]. 南京农业大学学报,2017,40(4):697-702. DOI:10.7685/jnau.201608021. Xu D,Wu X K,Jing Y W,et al. Catalytic activity of Brucella abortus SahH in activated methionine cycle[J]. Journal of Nanjing Agricultural University,2017,40(4):697-702(in Chinese with English abstract).
[12] Xu D,Zuo J,Chen Z,et al. Different activated methyl cycle pathways affect the pathogenicity of avian pathogenic Escherichia coli[J]. Veterinary Microbiology,2017,211:160-168.
[13] Han X,Lu C.Biological activity and identification of a peptide inhibitor of LuxS from Streptococcus suis serotype 2[J]. FEMS Microbiol Lett,2009,294:16-23.
[14] Chiang P K,Gordon R K,Tal J,et al. S-adenosylmethionine and methylation[J]. FASEB Journal:Official Publication of the Federation of American Societies for Experimental Biology,1996,10(4):471-480.
[15] Kitade Y,Kozaki A,Gotoh T,et al. Synthesis of S-adenosyl-L-homocysteine hydrolase inhibitors and their biological activities[J]. Nucleic Acids Symposium Series,1999,42:25-26.
[16] Vandenplas C,Guillerm D,Guillerm G. A new series of mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase from beef liver[J]. Nucleosides & Nucleotides,1999,18(4/5):569-570.
[17] Yamada T,Komoto J,Lou K,et al. Structure and function of eritadenine and its 3-deaza analogues:potent inhibitors of S-adenosylhomocysteine hydrolase and hypocholesterolemic agents[J]. Biochemical Pharmacology,2007,73(7):981-989.
[18] Bujnicki J M,Prigge S T,Caridha D,et al. Structure,evolution,and inhibitor interaction of S-adenosyl-L-homocysteine hydrolase from plasmodium falciparum[J]. Proteins,2003,52(4):624-632.
[19] Cai S,Li Q S,Fang J,et al. The rationale for targeting the NAD/NADH cofactor binding site of parasitic S-adenosyl-L-homocysteine hydrolase for the design of anti-parasitic drugs[J]. Nucleosides,Nucleotides & Nucleic Acids,2009,28(5):485-503.
[20] Singh D B,Dwivedi S. Structural insight into binding mode of inhibitor with SAHH of Plasmodium and human:interaction of curcumin with anti-malarial drug targets[J]. Journal of Chemical Biology,2016,9(4):107-120.
[21] Corrales R M,Leiba J,Cohen-Gonsaud M,et al. Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase is negatively regulated by Ser/Thr phosphorylation[J]. Biochemical and Biophysical Research Communications,2013,430(2):858-864.

相似文献/References:

[1]徐达,吴小卡,荆雅玮,等.布鲁菌半胱氨酸水解酶在甲硫氨酸循环中的催化活性研究[J].南京农业大学学报,2017,40(4):697.[doi:10.7685/jnau.201608021]
 XU Da,WU Xiaoka,JING Yawei,et al.Catalytic activity of Brucella abortus SahH in activated methionine cycle[J].Journal of Nanjing Agricultural University,2017,40(5):697.[doi:10.7685/jnau.201608021]

备注/Memo

备注/Memo:
收稿日期:2019-10-14。
基金项目:国家自然科学基金项目(31572546,31872483);国家重点研发计划专项(2018YFE0102200);上海市科技兴农重点攻关项目(2019-02-08-00-08-F01151)
作者简介:荆雅玮,硕士研究生。
通信作者:韩先干,研究员,博士,主要从事微生物致病机制、动物疫苗及动物疫病检测方法研究,E-mail:hanxgan@163.com。
更新日期/Last Update: 1900-01-01