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 μM 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" α-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.
Highlights
Quorum-quenching catalysts are of interest for potential application as biochemical tools to interrogate interbacterial communication pathways, as anti-biofouling agents, and as anti-infective agents in plants and animals
We used a purification procedure similar to that for other acyl-Lhomoserine lactones (AHLs) lactonases,[19] we found that purified AidC does not contain the typical two equivalents of zinc ions
The quorum-quenching AHL lactonase from the potato root-associated Chryseobacterium sp. strain StRB126, AidC, has an unusually low KM value for AHL substrates and displays a stricter substrate selectivity than any other related AHL lactonase characterized to date
Summary
Quorum-quenching catalysts are of interest for potential application as biochemical tools to interrogate interbacterial communication pathways, as anti-biofouling agents, and as anti-infective agents in plants and animals. The structures reveal two atypical features as compared to previously characterized AHL lactonases: a ‘kinked’ α-helix that forms part of a closed binding pocket which provides affinity and enforces selectivity for AHL substrates, and an active-site His substitution that is usually found in a homologous family of phosphodiesterases. 240B111) and AiiB (autoinducer inactivator B from Agrobacterium tumefaciens C5812) show only ~20% and ~17% amino acid identity, respectively These sequence alignments predict that active-site Asp and Tyr residues, conserved in other AHL lactonases due to their roles in zinc-binding and catalytic turnover, are possibly replaced in AidC by Leu and Ser, respectively.[9, 13, 14] the most striking feature of AidC is its reported KM values. Two aspects of the active site are atypical compared to previously characterized AHL lactonases: a novel substrate-binding pocket defined, in part, by an unusual ‘kinked’ αhelix containing an internal proline residue, and an active site His substitution that is usually found in more distant superfamily members that belong to a different family, the phosphodiester hydrolases
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