Purification, Characterization, and Stereochemical Analysis of a C−C Hydrolase:  2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic Acid 5,6-Hydrolase

Abstract

2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli has been purified to near homogeneity from an overexpressing strain of E. coli. The purified enzyme is a 29 kDa dimeric protein requiring no cofactors for catalytic activity. The enzyme has a Km of 2.1 microM and a kcat of 36 s-1 for its natural substrate and shows high selectivity for the propionate side chain of the substrate. The stereochemical course of the MhpC reaction was elucidated by conversion of protiosubstrate in 2H2O and conversion of deuteriated substrate in 1H2O, revealing that the reaction proceeds with overall replacement of a succinyl moiety by a proton from water in the H-5E position, with retention of regiochemistry. Isotope exchange was also observed in the H-5Z position of the product, which was rationalized by enzyme-catalyzed exchange of 2H into C-5 of the substrate from 2H2O. These data are consistent with a reversible keto-enol tautomerization taking place as the first step of the enzyme mechanism.

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

“…This species very slowly decayed (1/ 2 ϭ 0.0077 s Ϫ1 ) at a rate that matches the previously measured k cat value (Table 1). Interestingly, this rate is similar to that of tautomerization of HOPDA in solution as measured by deuterium exchange experiments (20,22). The absence of E⅐S red accumulation suggests that 3-Cl HOPDA tautomerization is slower than hydrolysis.…”

Hydrolytic Polyketide Shortening by Ayg1p, a Novel Enzyme Involved in Fungal Melanin Biosynthesis

“…Pseudomonas putida TodF (23) and XylF (24), Pseudomonas azelaica HbpD (25), Rhodococcus sp. EtbDs (26) and BphD (27), Pseudomonas fluorescens CumD (28, 29), and E. coli MhpC (30). Their substrates are ketonic ring fission products of aromatic compounds and are hydrolytically converted to smaller molecules.…”

Identification of a Serine Hydrolase as a Key Determinant in the Microbial Degradation of Polychlorinated Biphenyls

“…It has been proposed that MhpC, an enzyme related to BphD, binds a trans transoid enol tautomer of its substrate and catalyzes its tautomerization to the keto form through the action of an active site base and an active site acid (22,23). Base-catalyzed attack of the keto tautomer by water at C-6 yields a gem-diol intermediate prior to carbon-carbon bond fragmentation (21).…”

“…Interestingly, the conserved serine residue appears to act as a base (21). More specifically, mechanistic studies on MhpC, a hydrolase involved in the degradation of phenylpropionate, indicate that the substrate is first bound in the enol form and undergoes tautomerization to the keto form before being subject to base-catalyzed attack by water (21)(22)(23). It has been proposed that the keto form of the substrate binds weakly and can dissociate from the active site prior to attack by water.…”