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 t estosteroni strain B-356 is the second enzyme of the biphenyl/polychlorinat ed biphenyl degradation pathway. Based on the crystal structure of a relate d BphB, three conserved residues, Ser142, Tyr155, and Lys159, have been sug gested to function as a "catalytic triad" as for other members of the short -chain alcohol dehydrogenase/reductase (SDR) family. In this study, substit ution of each triad residue was examined in BphB. At pH 9.0, turnover numbe rs relative to wild-type enzyme were as follows: Y155F, 0.1%; S142A, 1%; an d 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 s howed Little dehydrogenase activity at pH 7, was sharply activated by incre asing the pH, reaching almost 25% of the activity of the wild-type enzyme a t pH 9.8. These three residues are therefore critical for BphB activity, as suggested by the crystal structure and similarity to other SDR family memb ers. In addition, BphB showed a strong preference for NAD(+) over NADP(+), with a 260-fold higher specificity constant (k(cat)/K-m). Evidence is prese nted that the inefficient use of NADP(+) by BphB might partly be due to the presence of an aspartate residue at position 36.