Document Detail

Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase.
MedLine Citation:
PMID:  23461395     Owner:  NLM     Status:  MEDLINE    
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, 40911-40920]. This enzyme possesses the (β/α)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 Å 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 determined to a resolution of 2.1 Å and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 Å, and the structures of ligand-free zinc-reconstituted wild-type, E101N, R230D, and E101G/R230C mutants of GKL were determined to resolutions of 2.1, 2.1, 1.9, and 2.0 Å, 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 512-553 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 α-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.
Bo Xue; Jeng Yeong Chow; Amgalanbaatar Baldansuren; Lai Lai Yap; Yunn Hwen Gan; Sergei A Dikanov; Robert C Robinson; Wen Shan Yew
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-03-19
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-04-02     Completed Date:  2013-05-21     Revised Date:  2014-04-04    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2359-70     Citation Subset:  IM    
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MeSH Terms
4-Butyrolactone / analogs & derivatives*,  metabolism
Acyl-Butyrolactones / metabolism
Amidohydrolases / chemistry*,  genetics,  metabolism*
Catalytic Domain
Crystallography, X-Ray
Geobacillus / chemistry,  enzymology*,  genetics,  metabolism
Manganese / metabolism
Models, Molecular
Quorum Sensing*
Grant Support
Reg. No./Substance:
0/Acyl-Butyrolactones; 0/N-butyrylhomoserine lactone; 42Z2K6ZL8P/Manganese; EC 3.5.-/Amidohydrolases; OL659KIY4X/4-Butyrolactone

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