title: Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase.
abstract: 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.
authors: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673.
authors: Bo
authors: B
authors: Xue
authors: Jeng Yeong
authors: JY
authors: Chow
authors: Amgalanbaatar
authors: A
authors: Baldansuren
authors: Lai Lai
authors: LL
authors: Yap
authors: Yunn Hwen
authors: YH
authors: Gan
authors: Sergei A
authors: SA
authors: Dikanov
authors: Robert C
authors: RC
authors: Robinson
authors: Wen Shan
authors: WS
authors: Yew
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D054742:Acyl-Butyrolactones / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D056506:Geobacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D053038:Quorum Sensing
chemical_list: D054742:Acyl-Butyrolactones; C092312:N-butyrylhomoserine lactone; D008345:Manganese; D000581:Amidohydrolases; D015107:4-Butyrolactone