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

doi:10.1002/prot.22009

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	Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori
	Wu, Dalei 1; Zhang, Liang 1; Kong, Yunhua 1; Du, Jiamu 2; Chen, Shuai 1; Chen, Jing1 ; Ding, Jianping 2; Jiang, Hualiang1,3 ; Shen, Xu1,3
刊名	PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
	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
DOI	10.1002/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.
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
WOS研究方向	Biochemistry & Molecular Biology ; Biophysics
语种	英语
出版者	WILEY
WOS记录号	WOS:000259287500005
内容类型	期刊论文
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.