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Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

文献类型：期刊论文

作者Wu, Dalei1; Zhang, Liang1; Kong, Yunhua1; Du, Jiamu2; Chen, Shuai1; Chen, Jing1; Ding, Jianping2; Jiang, Hualiang1,3; Shen, Xu1,3
刊名PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
出版日期2008-09
卷号72期号:4页码:1148-1160
关键词D-alanine-D-alanine ligase Helicobacter pylori crystal structure complementary assay mutant peptidoglycan biosynthesis pathway
ISSN号0887-3585
DOI10.1002/prot.22009
文献子类Article
英文摘要D-Alanine-D-alanine ligase is tire second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SSI (HpDdl) was kinetically and structurally characterized. The determined apparent K of ATP (0.87 mu M), the K-ml (1.89 mM) and K-m2 of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against tire substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of fire D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of fire enzyme. Kinetic analyses against tire corresponding HpDdl mutants (16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of tire enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in tire C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of tire enzyme. Our present work hopefully provides useful information for understanding tire D-Ala-D-Ala ligase of Helicobacter pylori.
WOS关键词ALANYL-D-ALANINE ; RESISTANT LEUCONOSTOC-MESENTEROIDES ; ACTIVE-SITE MUTANTS ; D-LACTATE LIGASE ; VANCOMYCIN RESISTANCE ; ESCHERICHIA-COLI ; CELL-WALL ; D-CYCLOSERINE ; GENE CLONING ; DDLA GENE
资助项目State Key Program of Basic Research of China[2004CB58905] ; State Key Program of Basic Research of China[2007CB914304] ; State Key Program of Basic Research of China[2006AA09Z447] ; National Natulral Science Foundation of China[30525024] ; National Natulral Science Foundation of China[90713046] ; National Natulral Science Foundation of China[20721003] ; Shanghai Science and Technology Commission[06JC14080] ; Shanghai Science and Technology Commission[03DZ191228] ; Foundation of Chinese Academy of Sciences[KSCX1-YW- R-18]
WOS研究方向Biochemistry & Molecular Biology ; Biophysics
语种英语
WOS记录号WOS:000259287500005
出版者WILEY
源URL[http://119.78.100.183/handle/2S10ELR8/272829]  
专题药物发现与设计中心
中科院受体结构与功能重点实验室
新药研究国家重点实验室
药理学第三研究室
通讯作者Shen, Xu
作者单位1.Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Drug Discovery & Design Ctr, Shanghai 201203, Peoples R China;
2.Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Biochem & Cell Biol, State Key Lab Mol Biol, Shanghai 200031, Peoples R China;
3.E China Univ Sci & Technol, Sch Pharm, Shanghai 200237, Peoples R China
推荐引用方式
GB/T 7714		 Wu, Dalei,Zhang, Liang,Kong, Yunhua,et al. Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori[J]. PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS,2008,72(4):1148-1160.
APA Wu, Dalei.,Zhang, Liang.,Kong, Yunhua.,Du, Jiamu.,Chen, Shuai.,...&Shen, Xu.(2008).Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori.PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS,72(4),1148-1160.
MLA Wu, Dalei,et al."Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori".PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 72.4(2008):1148-1160.

入库方式： OAI收割

来源：上海药物研究所

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