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 time 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 for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 ؎ 16 M and 46 ؎ 4 nmol min ؊1 g ؊1 , respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.

Nucleotide sequence of the gene encoding cis-biphenyl dihydrodiol dehydrogenase (bphB) and the expression of an active recombinant His-tagged bphB gene product from a PCB degrading bacterium, Pseudomonas putida OU83

“…The preferential usage of G and C‐terminated codons contributes to the relatively high G+C content, characteristic of many pseudomonads [20]. The termination codon of the bphB gene, TGA, has been reported to be in the bphB gene of Comamonas testosteroni strain B‐356 [5]. However, in most other biphenyl degrading strains, Pseudomonas sp.…”

“…The BDDH activity was measured by the method of Sylvestre et al [5]. Protein concentration was determined using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) [17].…”

“…The product, cis ‐biphenyl‐2,3‐dihydrodiol, is oxidized to 2,3‐dihydroxybiphenyl by cis ‐biphenyl dihydrodiol dehydrogenase (BDDH), encoded by the bphB gene (Fig. 1) [1–5]. 2,3‐Dihydroxybiphenyl is cleaved by an extradiol meta ‐cleaving dioxygenase, 2,3‐dihydroxybiphenyl‐1,2‐dioxygenase, encoded by the bphC gene.…”

Cloning and characterization of a novel cis-naphthalene dihydrodiol dehydrogenase gene (narB) from Rhodococcus sp. NCIMB12038

“…Earlier alignments of the amino acid sequences of bac- ; NahB BS202, NahB of P. putida BS202, naphthalene metabolism (AF010471); NahB G7, NahB of P. putida G7, naphthalene metabolism (AF125184); NahB AN10, NahB of P. stutzeri AN10, naphthalene metabolism (AF039533); NagB of Pseudomonas sp. U2, naphthalene metabolism (AF036940); terial aryl-dihydrodiol dehydrogenases showed the existence of two classes of these enzymes [23]. The ¢rst class comprises enzymes involved in the degradation of monoaryls with a carboxyl group (e.g.…”

“…One cluster includes dehydrogenases involved in the degradation of compounds for which the initial reaction is catalyzed by class II dioxygenases (e.g. biphenyl 2,3-dioxygenase) and the other by class III dioxygenases (naphthalene 1,2-dioxygenase) [4,23]. To reveal the evolutionary relationship between NarB and the other dehydrogenases we carried out a phylogenetic analysis.…”