Characterization of active recombinant 2,3-dihydro-2,3- dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB)

Abstract

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first lime some of the main features of a B2,3D. This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The K(m) value and maximum rate of metabolism fur 2,3-dihydro-2,3-dihydroxybiphenyl were 73 ± 16 μM and 46 ± 4 nmol min-1 μg-1, respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be inure substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.