Abstract

DmpFG, a bifunctional aldolase/dehydrogenase (acylating), comprises the last two enzymes of the (dimethyl)phenol degradation pathway in Pseudomonas sp. strain CF600. DmpG, a class II divalent cation dependent aldolase belonging to the HMGL-like family, cleaves 4-hydroxy-2-ketovalerate to pyruvate and acetaldehyde while DmpF, an NAD+ /CoA dependent aldehyde dehydrogenase (acylating), oxidizes the acetaldehyde to acetyl-CoA. Studies of the DmpF active site focused on delineation of the coenzyme A binding site, and involvement of a conserved cysteine residue, Cys132, in catalysis and substrate-mediated inactivation. By coupling hydrogen-deuterium exchange experiments with peptic digest/mass spectrometric analysis, coenzyme A was shown to share the same binding site as NAD + . Cys132 was identified as an essential cysteine, and was shown to form an acetyl-Cys132 intermediate which was trapped and identified by mass spectrometry. Coenzyme A-mediated inactivation resulted from disulfide bond formation between thiol groups of Cys132 and coenzyme A. Loss of activity in the presence of acetaldehyde was incurred by the oxidation of Cys132 to cysteine sulfinic acid, as observed by mass spectrometry. Studies of DmpG focused on the roles of metal ions and putative active site residues. Purified preparations contained a mixture of Fe, Mn, Zn and Cu, in order of decreasing content, with one mol total metal per mol protein. However, this tightly-bound metal could only support residual adolase activity, with full activity requiring high concentrations of Mn 2+ or Co2+ in the assay, suggesting two classes of metal binding sites. Site-directed mutagenesis was carried out on the three ligands to the tightly bound metal ion, Asp18, His200 and His202, as well as another three residues, potentially involved in catalysis, Arg17, His21 and Tyr291. D18N, H200A and H202A variants were depleted of bound metals, concomitant with complete loss of the residual aldolase activity. In the presence of Mn 2+ or Co2+ , aldolase activities of D18N and H200A variants remained very low in comparison with WT, but H202A aldolase activity was comparable to WT. Substitutions of Arg17 or His21 significantly impaired aldolase activity, while Tyr291 variants had activity similar to WT. Properties of these variants are discussed in relation to the family of HMGL-like enzymes.