4LXI

Crystal Structure of the S105A mutant of a carbon-carbon bond hydrolase, DxnB2 from Sphingomonas wittichii RW1, in complex with 5,8-diF HOPDA

Summary for 4LXI

• Entry DOI: 10.2210/pdb4lxi/pdb
• Related: 4LXG 4LXH
• Descriptor: MCP Hydrolase, (3E,5R)-5-fluoro-6-(2-fluorophenyl)-2,6-dioxohex-3-enoic acid, SODIUM ION, ... (4 entities in total)
• Functional Keywords: carbon-carbon bond hydrolase, rossmann fold, alpha/beta hydrolase fold, carbon-carbon bond hydrolysis, hydrolase
• Biological source: Sphingomonas wittichii RW1
• Total number of polymer chains: 1
• Total formula weight: 30485.71

Authors

Bhowmik, S.,Bolin, J.T. (deposition date: 2013-07-29, release date: 2013-10-09, Last modification date: 2024-02-28)

Primary citation

Ruzzini, A.C.,Bhowmik, S.,Ghosh, S.,Yam, K.C.,Bolin, J.T.,Eltis, L.D.
A substrate-assisted mechanism of nucleophile activation in a ser-his-asp containing C-C bond hydrolase.
Biochemistry, 52:7428-7438, 2013

PubMed Abstract: The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (βnuc ∼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

PubMed: 24067021
DOI: 10.1021/bi401156a

Experimental method

X-RAY DIFFRACTION (2.17 Å)