4h9v
Structure of Geobacillus kaustophilus lactonase, mutant E101G/R230C with Zn2+
Structural highlights
FunctionPublication Abstract from PubMedThe 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 (beta/alpha)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 A 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 A and the structure of the ligand-free, manganese-reconstituted E101N mutant to a resolution of 2.6 A, 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 A, 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 alpha-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. Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase.,Xue B, Chow JY, Baldansuren A, Yap LL, Gan YH, Dikanov SA, Robinson RC, Yew WS Biochemistry. 2013 Apr 2;52(13):2359-70. doi: 10.1021/bi4000904. Epub 2013 Mar, 19. PMID:23461395[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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