2ri6

From Proteopedia

Revision as of 10:24, 23 January 2008

Crystal Structure of S112A mutant of a C-C hydrolase, BphD from Burkholderia xenovorans LB400

Overview

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond, hydrolysis of 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid (HOPDA) to, afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An, enol-keto tautomerization has been proposed to precede hydrolysis via a, gem-diol intermediate. The role of the canonical 'catalytic triad', (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains, unclear. We previously reported that the BphD-catalyzed hydrolysis of, HOPDA (max = 434 nm for the free enolate) proceeds via an unidentified, intermediate with a red-shifted absorption spectrum (max = 492 nm), (Horsman et al. (2006), Biochemistry 45, 11071). Here we demonstrate that, the Ser112Ala variant (S112A) generates and traps a similar intermediate, (max = 506 nm) with a similar rate, 1/t ~ 500 s-1. The crystal structure, of the S112A:HOPDA complex at 1.8 A resolution identified this, intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate., This keto tautomer did not accumulate in either the H265A or the, S112A/H265A double variants, indicating that His-265 catalyzes, tautomerization. Consistent with this role, the wild type and S112A, enzymes catalyzed tautomerization of the product HPD, while H265A variants, did not. This study thus identifies a keto intermediate, and demonstrates, that the catalytic triad histidine catalyzes the tautomerization, half-reaction, expanding the role of this residue from its purely, hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA, crystal structure is more consistent with hydrolysis occurring via an, acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules, have poor access to C6, and the closest ordered water is 7 A away.

About this Structure

2RI6 is a Single protein structure of sequence from Burkholderia xenovorans with and as ligands. This structure superseeds the now removed PDB entry 2PU6. Full crystallographic information is available from OCA.

Reference

The tautomeric half-reaction of BphD, A C-C bond hydrolase: Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad., Horsman GP, Bhowmik S, Seah SY, Kumar P, Bolin JT, Eltis LD, J Biol Chem. 2007 Apr 18;. PMID:17442675

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