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Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.

ABSTRACT: Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a kcat/KM value of approximately 2 × 10(6) M(-1) s(-1) for N-heptanoyl-l-homoserine lactone. The enzyme has stricter substrate selectivity and significantly lower KM values (ca. 50 ?M for preferred substrates) compared to those of typical AHL lactonases (ca. >1 mM). X-ray crystal structures of AidC alone and with the product N-hexanoyl-l-homoserine were determined at resolutions of 1.09 and 1.67 Å, respectively. Each structure displays as a dimer, and dimeric oligiomerization was also observed in solution by size-exclusion chromatography coupled with multiangle light scattering. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a "kinked" ?-helix that forms part of a closed binding pocket that provides affinity and enforces selectivity for AHL substrates and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. Implications for the catalytic mechanism of AHL lactonases are discussed.

SUBMITTER: Mascarenhas R

PROVIDER: S-EPMC4681436 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.

Mascarenhas Romila R Thomas Pei W PW Wu Chun-Xiang CX Nocek Boguslaw P BP Hoang Quyen Q QQ Liu Dali D Fast Walter W

Biochemistry 20150708 28

Quorum-quenching catalysts are of interest for potential application as biochemical tools for interrogating interbacterial communication pathways, as antibiofouling agents, and as anti-infective agents in plants and animals. Herein, the structure and function of AidC, an N-acyl-l-homoserine lactone (AHL) lactonase from Chryseobacterium, is characterized. Steady-state kinetics show that zinc-supplemented AidC is the most efficient wild-type quorum-quenching enzymes characterized to date, with a k ...[more]

PMID: 26115006

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Project description: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 (?/?)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 ?-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.

Dataset Information

Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.

Publications

Structural and Biochemical Characterization of AidC, a Quorum-Quenching Lactonase with Atypical Selectivity.

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