2pu7
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Crystal Structure of S112A/H265A double mutant of a C-C hydrolase, BphD, from Burkholderia xenovorans LB400
OverviewOverview
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 StructureAbout this Structure
2PU7 is a Single protein structure of sequence from Burkholderia xenovorans with NA and MLI as ligands. Full crystallographic information is available from OCA.
ReferenceReference
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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