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The purpose of this investigation was to examine the capacity of the biphenyl catabolic enzymes of Comamonas testosteroni B-356 to metabolize dihydroxybiphenyls symmetrically substituted on both rings. Data show that 3,3'-dihydroxybiphenyl is by far the preferred substrate for strain B-356. However, the dihydrodiol metabolite is very unstable and readily tautomerizes to a dead-end metabolite or is dehydroxylated by elimination of water. The tautomerization route is the most prominent. Thus, a very small fraction of the substrate is converted to other hydroxylated and acidic metabolites. Although 2,2'-dihydroxybiphenyl is a poor substrate for strain B-356 biphenyl dioxygenase, metabolites were produced by the biphenyl catabolic enzymes, leading to production of 2-hydroxybenzoic acid. Data show that the major route of metabolism involves, as a first step, a direct dehydroxylation of one of the ortho-substituted carbons to yield 2,3,2'-trihydroxybiphenyl. However, other metabolites resulting from hydroxylation of carbons 5 and 6 of 2,2'-dihydroxybiphenyl were also produced, leading to dead-end metabolites. [ABSTRACT FROM AUTHOR]

The bacterial degradation of polychlorinated biphenyls depends on the ability of the enzyme biphenyl 2,3-dioxygenase (BPDO) to catalyze their oxygenation. Analysis of hybrid BPDOs obtained using common restriction sites to exchange large DNA fragments between LB400 bphA and B-356 bphA showed that the C-terminal portion of LB400 α subunit can withstand extensive structural modifications, and that these modifications can change the catalytic properties of the enzyme. On the other hand, exchanging the C-terminal portion of B-356 BPDO α subunit with that of LB400 α subunit generated inactive chimeras. Data encourage an enzyme engineering approach, consisting of introducing extensive modifications of the C-terminal portion of LB400 bphA to extend BPDO catalytic properties toward polychlorinated biphenyls.Key words: PCB, protein engineering, BphA, BPDO, polychlorinated biphenyl.La dégradation bactérienne des biphényles polychlorés dépend de la capacité de l'enzyme biphényle 2,3-dioxygénase (BPDO) à catalyser leur oxygénation. L'analyse de BPDO hybrides obtenues en utilisant des sites de restriction communs pour échanger de larges fragments d'ADN entre les gènes bphA de LB400 et de B-356, a montré que la sous unité α de LB400 restait active en dépit de modifications structurelles majeures dans sa portion C-terminale. De plus, ces modifications pouvaient changer les propriétés catalytiques de l'enzyme envers les BPC. Toutefois, le remplacement de la partie C-terminale de la sous unité α de B-356 par celle de LB400 a généré des enzymes hybrides inactives. Nos résultats nous amènent à proposer une approche pour l'ingénierie d'une BPDO potentiel d'activité accrue envers les biphényles polychlorés qui consisterait à introduire de façon aléatoire, d'importantes modifications dans la partie de bphA de LB400 qui code pour la portion C-terminale de la protéine.Mots clés : BPC, ingénierie des protéines, BphA, BPDO, biphényles polychlorés. [ABSTRACT FROM AUTHOR]

Examines the role of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) active site residues from Camamonas testosteroni in the bacterial degradation of polychlorinated biphenyls. Expression and purification of BphB; Structural basis for the nucleotide specificity of BphB; Analysis on relative activities of hydrogen ion concentration-rate reactions.