The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase

Abstract

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-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- Bacteria use meta-cleavage pathways to degrade a large variety of aromatic compounds, and some alicyclic compounds, such as steroids (1). Although each pathway has its own substrate specificity, they all employ the same underlying logic: vicinal dihydroxylation of an aromatic ring enables dioxygenase-catalyzed extradiol (or meta) ring opening. The resulting meta-cleavage product (MCP) 3 is degraded by an MCP hydrolase, which adds water across a carbon-carbon bond to generate a dienoate and a carboxylic acid (Fig.

The Molecular Basis for Inhibition of BphD, a C-C Bond Hydrolase Involved in Polychlorinated Biphenyls Degradation

“…The 4,4Ј-diF-DHB was dissolved in 10% ethanol, 80% H 2 O, and 10% D 2 O to collect NMR spectra at 25°C using a 600-MHz spectrometer at the Department of Chemistry, University of Rochester. The 1 H NMR reference compound was 100 M 2,2-dimethyl-2-silapentane 5-sulfonate and was used as an indirect reference for 19 25.0 Ϯ 0.5°C, controlled by Cary WinUV software version 2.00. Reactions were carried out in a 1-ml volume and were initiated by the addition of 5 l of an appropriately diluted enzyme solution.…”

“…Crystallization and Preparation of Complexes-Crystals of the substrate-free S112A variant of BphD were grown at 20°C in 1.9 M sodium malonate, pH 7.0, by sitting drop vapor diffusion, as previously reported (19). Complexes were prepared by incubating crystals for 30 min in 60 l of reservoir solution supplemented with ϳ10 mM 3-Cl-HOPDA or ϳ65 mM 3,10-difluoro-HOPDA.…”

“…Crystallographic data for the E⅐S red intermediate trapped in the S112A⅐HOPDA complex are most consistent with the bound HOPDA being ketonized (19), although a non-planar, distorted conformation of the enol/enolate could not be completely ruled out. Hydrolysis then proceeds via either a gem-diol intermediate (24 -28) or an acyl-enzyme intermediate (19,20). As noted above, HOPDAs that are chlorinated on the dienoate moiety are poorly transformed by BphD (8 -10).…”

“…To expel the electron-rich dienoate moiety of the substrate, the enzyme first employs a His-265-mediated enol-keto tautomerization to generate a hydrolyzable keto-intermediate (E⅐S k ) (19 -24). Indeed, His-265-dependent formation of an intermediate with a red-shifted absorbance spectrum (E⅐S red ) is formed at a similar rate in both WT and S112A BphDs (19). Crystallographic data for the E⅐S red intermediate trapped in the S112A⅐HOPDA complex are most consistent with the bound HOPDA being ketonized (19), although a non-planar, distorted conformation of the enol/enolate could not be completely ruled out.…”

“…Indeed, His-265-dependent formation of an intermediate with a red-shifted absorbance spectrum (E⅐S red ) is formed at a similar rate in both WT and S112A BphDs (19). Crystallographic data for the E⅐S red intermediate trapped in the S112A⅐HOPDA complex are most consistent with the bound HOPDA being ketonized (19), although a non-planar, distorted conformation of the enol/enolate could not be completely ruled out. Hydrolysis then proceeds via either a gem-diol intermediate (24 -28) or an acyl-enzyme intermediate (19,20).…”