CHARACTERIZATION OF ACTIVE RECOMBINANT 2,3-DIHYDRO-2,3-DIHYDROXYBIPHENYL DEHYDROGENASE FROM COMAMONAS TESTOSTERONI B-356 AND SEQUENCE OF THE ENCODING GENE (BPHB)

,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes t he second step in the biphenyl degradation pathway. The nucleotide seq uence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was d etermined, Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to eac h 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 pro duce recombinant active His-tagged B2,3D, which allowed us to describe for the first rime 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 ve ry similar to those reported for the related enzyme cis-toluene dihydr odiol dehydrogenase. The K-m value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 15 mu M and 46 +/- 4 nmo l min(-1) mu g(-1), respectively. Compared with the cis-toluene dihydr odiol dehydrogenase, B2,3D appeared to be more substrate specific sinc e it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.