Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356.
cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) from Comamonas testosteroni strain B-356 is the second enzyme of the biphenyl/polychlorinated biphenyl degradation pathway. Based on the crystal structure of a related BphB, three conserved residues, Ser142, Tyr155, and Lys159, have been suggested to function as a "catalytic triad" as for other members of the short-chain alcohol dehydrogenase/reductase (SDR) family. In this study, substitution of each triad residue was examined in BphB. At pH 9.0, turnover numbers relative to wild-type enzyme were as follows: Y155F, 0.1%; S142A, 1%; and K159A, 10%. Although the Michaelis constants of K159A and S142A for cis-2,3-dihydro-2,3-dihydroxybiphenyl increased about 20-fold, relatively little change was observed in the K(m) for dinucleotide. The K159A mutant, which showed little dehydrogenase activity at pH 7, was sharply activated by increasing the pH, reaching almost 25% of the activity of the wild-type enzyme at pH 9. 8. These three residues are therefore critical for BphB activity, as suggested by the crystal structure and similarity to other SDR family members. In addition, BphB showed a strong preference for NAD(+) over NADP(+), with a 260-fold higher specificity constant (k(cat)/K(m)). Evidence is presented that the inefficient use of NADP(+) by BphB might partly be due to the presence of an aspartate residue at position 36.
Vedadi
M
M
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada H3G 1M8.
Barriault
D
D
Sylvestre
M
M
Powlowski
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D020604:Comamonas testosteroni / Q000201:enzymology*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010088:Oxidoreductases; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase