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
文献类型：期刊论文
| 作者 | Wu, DL; Hu, TC; Zhang, L; Chen, J; Du, J; Ding, JP; Jiang, HL; Shen, X |
| 刊名 | PROTEIN SCIENCE
 |
| 出版日期 | 2008 |
| 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 |
| 通讯作者 | Jiang, HL (reprint author), Chinese Acad Sci, Shanghai Inst Mat Med, Drug Discovery & Design Ctr, State Key Lab Drug Res, Shanghai 201203, Peoples R China.,hljiang@mail.shcnc.ac.cn |
| 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. |
| 学科主题 | Biochemistry & Molecular Biology |
| 类目[WOS] | Biochemistry & Molecular Biology |
| 关键词[WOS] | BACILLUS-STEAROTHERMOPHILUS ; PSEUDOMONAS-AERUGINOSA ; PYRIDOXAL 5'-PHOSPHATE ; SALMONELLA-TYPHIMURIUM ; ANTIBIOTIC-RESISTANCE ; LYSINE 39 ; GENE ; BIOSYNTHESIS ; CYCLOSERINE ; MECHANISM |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000256166600011 |
| 版本 | 出版稿 |
| 源URL | [http://202.127.25.143/handle/331003/1398]  |
| 专题 | 上海生化细胞研究所_上海生科院生化细胞研究所 |
| 推荐引用方式 GB/T 7714 | Wu, DL,Hu, TC,Zhang, L,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, DL.,Hu, TC.,Zhang, L.,Chen, J.,Du, J.,...&Shen, X.(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, DL,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. |
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