Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E-coli: Enzymatic characterization with crystal structure analysis

doi:10.1110/ps.083495908

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	Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E-coli: Enzymatic characterization with crystal structure analysis
	Wu, Dalei 1; Hu, Tiancen 1; Zhang, Liang 1; Chen, Jing1 ; Du, Jiamu 2; Ding, Jianping 2; Jiang, Hualiang1 ; Shen, Xu1
刊名	PROTEIN SCIENCE
	2008-06
卷号	17 期号:6 页码:1066-1076
关键词	alanine racemase substrate entryway crystal structure Escherichia coli pyridoxal 5'-phosphate charged residues
ISSN号	0961-8368
DOI	10.1110/ps.083495908
文献子类	Article
英文摘要	Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D-to L-Ala and L-to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.
WOS关键词	BACILLUS-STEAROTHERMOPHILUS ; PSEUDOMONAS-AERUGINOSA ; PYRIDOXAL 5'-PHOSPHATE ; SALMONELLA-TYPHIMURIUM ; ANTIBIOTIC-RESISTANCE ; LYSINE 39 ; GENE ; BIOSYNTHESIS ; CYCLOSERINE ; MECHANISM
WOS研究方向	Biochemistry & Molecular Biology
语种	英语
出版者	WILEY
WOS记录号	WOS:000256166600011
内容类型	期刊论文
源URL	[http://119.78.100.183/handle/2S10ELR8/272913]
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
通讯作者	Jiang, Hualiang
作者单位	1.Chinese Acad Sci, Shanghai Inst Mat Med, Drug Discovery & Design Ctr, State Key Lab Drug Res, 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
推荐引用方式 GB/T 7714	Wu, Dalei,Hu, Tiancen,Zhang, Liang,et al. Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E-coli: Enzymatic characterization with crystal structure analysis[J]. PROTEIN SCIENCE,2008,17(6):1066-1076.
APA	Wu, Dalei.,Hu, Tiancen.,Zhang, Liang.,Chen, Jing.,Du, Jiamu.,...&Shen, Xu.(2008).Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E-coli: Enzymatic characterization with crystal structure analysis.PROTEIN SCIENCE,17(6),1066-1076.
MLA	Wu, Dalei,et al."Residues Asp164 and Glu165 at the substrate entryway function potently in substrate orientation of alanine racemase from E-coli: Enzymatic characterization with crystal structure analysis".PROTEIN SCIENCE 17.6(2008):1066-1076.