| Title | Catalytic Mechanism of SHCHC Synthase in the Menaquinone Biosynthesis of Escherichia coli: Identification and Mutational Analysis of the Active Site Residues |
| Authors | Jiang, Ming Chen, Xiaolei Wu, Xian-Hui Chen, Minjiao Wu, Yun-Dong Guo, Zhihong |
| Affiliation | Hong Kong Univ Sci & Technol, Dept Chem, Ctr Canc Res, Kowloon, Hong Kong, Peoples R China. Peking Univ, Lab Chem Genom, Shenzhen Grad Sch, Shenzhen, Peoples R China. |
| Keywords | O-SUCCINYLBENZOATE SYNTHASE ACYLAMINO ACID RACEMASE ONE-PROTON CATALYSIS SERINE PROTEASES CROTONASE SUPERFAMILY DIRECTED MUTAGENESIS DIVERGENT EVOLUTION ENOLASE SUPERFAMILY ALPHA-CHYMOTRYPSIN CRYSTAL-STRUCTURE |
| Issue Date | 2009 |
| Publisher | biochemistry us |
| Citation | BIOCHEMISTRY.2009,48,(29),6921-6931. |
| Abstract | (1R,6R)-2-Succinyl-6-liydi-oxy-2,4-cyclohexadiene-l-carboxylate(SHCHC) synthase(MenH) is an alpha/beta fold enzyme containing a catalytically essential serine-histidine-aspartate triad typical of serine proteases but catalyzes a pyruvate elimination reaction initiated by alpha-proton abstraction in the menaquinone blosynthetic pathway of Escherichia coli. In this study, we identify the active site residues in the synthase through sequence analysis and structural modeling and study their mechanistic roles in MenH catalysis. Steady-state kinetic characterization of site-directed mutants of the active site residues shows that three conserved arginine residues (Arg-90, Arg-124, and Arg-168) likely form ionic salt bridges with three carboxylate groups of the substrate in the Michaelis complex and that the side-chain polar groups of the conserved tyrosine (Tyr-85) and tryptophan (Trp-147) residues likely donate hydrogen bonds to form all "oxyanion hole". In addition, the pH dependence of the MenH kinetic properties reveals a catalytic base with a pK(a) highly dependent on the hydroxyl group of the triad serine residue in the enzymatic reaction. Moreover, proton inventory experiments demonstrate that the SHCHC synthase adopts one-proton catalysis like many serine proteases. These results allow the proposal of a mechanism in which the histidine residue of the MenH triad serves as a general base catalyst to deprotonate the triad seryl hydroxyl group in the alpha-prolon abstraction from the substrate. As Such, the MenH triad performs a simple and fundamental proton transfer reaction occurring repeatedly in the reactions catalyzed by serine proteases and alpha/beta fold hydrolases, Suggesting a common evolutionary origin for all serine-histidine-aspartate triads serving different catalytic functions. |
| URI | http://hdl.handle.net/20.500.11897/246795 |
| ISSN | 0006-2960 |
| DOI | 10.1021/bi900897h |
| Indexed | SCI(E) EI |
| Appears in Collections: | 深圳研究生院待认领 |
