Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.
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 beta-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 logV and logV/K pH-rate profiles for benzaldehyde with either NAD+ or NADP+ as the coenzyme are both bell-shaped. The pKa 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 pKa of Cys249 was 8.4, i.e., Cys249 is not responsible for the pKas observed in the pH-rate profiles.
Yeung
Catherine K
CK
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, MI 48109, USA.
Yep
Alejandra
A
Kenyon
George L
GL
McLeish
Michael J
MJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D050727:Benzaldehyde Dehydrogenase (NADP+) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D050727:Benzaldehyde Dehydrogenase (NADP+)