Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

Abstract

The mandelate pathway of Pseudomonas putida ATCC 12633 comprises five enzymes and catalyzes the conversion of R- and S-mandelamide to benzoic acid which subsequently enters the β-ketoadipate pathway. Although the first four enzymes have been extensively characterized the terminal enzyme, a NAD(P) +-dependent benzaldehyde dehydrogenase (BADH), remains largely undescribed. Here we report that BADH is a dimer in solution, and that DTT is necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme. Site-directed mutagenesis confirms that Cys249 is the catalytic cysteine and identifies Cys140 as the cysteine likely to be involved in inter-monomer disulfide formation. BADH can utilize a range of aromatic substrates and will also operate efficiently with cyclohexanal as well as medium-chain aliphatic aldehydes. The log V and log V/ K pH-rate profiles for benzaldehyde with either NAD + or NADP + as the coenzyme are both bell-shaped. The p K a values on the ascending limb range from 6.2 to 7.1 while those on the descending limb range from 9.6 to 9.9. A spectrophotometric approach was used to show that the p K a of Cys249 was 8.4, i.e., Cys249 is not responsible for the p K as observed in the pH-rate profiles.