Structural Evidence of a Productive Active Site Architecture for an Evolved Quorum-Quenching GKL Lactonase

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• 作者：Bo Xue ; Jeng Yeong Chow ; Amgalanbaatar Baldansuren ; Lai Lai Yap ; Yunn Hwen Gan ; Sergei A. Dikanov ; Robert C. Robinson ; Wen Shan Yew
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：April 2, 2013
• 年：2013
• 卷：52
• 期：13
• 页码：2359-2370
• 全文大小：506K
• 年卷期：v.52,no.13(April 2, 2013)
• ISSN：1520-4995

文摘

The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as a template, yielding the E101G/R230C GKL mutant with increased catalytic activity and a broadened substrate range [Chow, J. Y., Xue, B., Lee, K. H., Tung, A., Wu, L., Robinson, R. C., and Yew, W. S. (2010) J. Biol. Chem. 285, 40911鈥?0920]. This enzyme possesses the (尾/伪)₈-barrel fold and is a member of the PLL (phosphotriesterase-like lactonase) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acyl-homoserine lactones, which mediate the quorum-sensing pathways of bacteria. The structure of the evolved N-butyryl-l-homoserine lactone (substrate)-bound E101G/R230C GKL enzyme was determined, in the presence of the inactivating D266N mutation, to a resolution of 2.2 脜 to provide an explanation for the observed rate enhancements. In addition, the substrate-bound structure of the catalytically inactive E101N/D266N mutant of the manganese-reconstituted enzyme was determinied to a resolution of 2.1 脜 and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 脜, and the structures of ligand-free zinc-reconstituted wild-type, E101N, R230D, and E101G/R230C mutants of GKL were determinied to resolutions of 2.1, 2.1, 1.9, and 2.0 脜, respectively. In particular, the structure of the evolved E101G/R230C mutant of GKL provides evidence of a catalytically productive active site architecture that contributes to the observed enhancement of catalysis. At high concentrations, wild-type and mutant GKL enzymes are differentially colored, with absorbance maxima in the range of 512鈥?53 nm. The structures of the wild-type and mutant GKL provide a tractable link between the origins of the coloration and the charge-transfer complex between the 伪-cation and Tyr99 within the enzyme active site. Taken together, this study provides evidence of the modulability of enzymatic catalysis through subtle changes in enzyme active site architecture.