4LYE

Crystal structure of the S105A mutant of a C-C hydrolase, DxnB2 from Sphingomonas wittichii RW1, in complex with substrate HOPDA

Summary for 4LYE

• Entry DOI: 10.2210/pdb4lye/pdb
• Related: 4LXG 4LXH 4LXI 4LYD
• Descriptor: MCP Hydrolase, (3E)-2,6-DIOXO-6-PHENYLHEX-3-ENOATE (3 entities in total)
• Functional Keywords: meta-cleavage product hydrolase, c-c bond hydrolase, alpha-beta hydrolase, dibenzo-p-dioxin degradation, dibenzofuran degradation, hydrolase
• Biological source: Sphingomonas wittichii
• Total number of polymer chains: 1
• Total formula weight: 30425.73

Authors

Ghosh, S.,Bolin, J.T. (deposition date: 2013-07-31, 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 (2.33 Å)