Characterization of the molecular basis for polychlorinated biphenyls (PCBs) transformation by biphenyl dioxygenase

Biphenyl dioxygenase (BPDO) is the first enzyme in the bph pathway, catalyzing the dihydroxylation of biphenyl and some polychlorinated biphenyls. The BPDOs of different bacterial strains possess different abilities to transform PCBs. To better understand the molecular basis of these different abilities, highly active preparations of four BPDOs were anaerobically purified and characterized: BPDO[sub LB400] from Burkholderia xenovorans LB400, BPDO[sub B356] from Comamonas testosteroni B-356, and two engineered variants, BPDO₁₁₉ and BPDO₁₁₁₀. BPDO₁₁₉ is a variant of BPDO[sub LB400] containing four substituted residues: T335G, F336I, N338T, and I341T. These residues correspond to those found in BPDO[sub B356], belong to region III identified by Mondello et al, and contribute to the substrate-binding pocket of the enzyme. BPDO₁₁₁₀ contains these substitutions as well as A267S. Steady-state kinetics assays demonstrated that of the four variants, BPDO[sub B356] had the highest apparent k[sub cat] for biphenyl, which was 10-fold higher than that of BPDO[sub LB400]. By contrast, BPDO[sub LB400] had the highest apparent substrate specificity for biphenyl and was 10-fold higher than that of BPDO[sub B356]. The steady-state parameters of BPDO₁₁₉ and BPDO₁₁₁₀ for biphenyl were intermediate between those of the two parental enzymes. The identity of the residue at position 267 had a greater effect on the parameters than the identity o f the region III residues. In all variants, the consumption of oxygen was well-coupled to that of biphenyl. The abilities o f the four variant BPDOs to transform PCB congeners were investigated using three different methods: (1) purified enzymes and individual congeners; (2) purified enzymes and a mixture of 8 congeners; and (3) whole cells and a mixture of 8 congeners. The results obtained by each method were consistent. Most strikingly, BPDO[sub B356] transformed a greater number of congeners at a faster rate than the other enzymes. Previously unrecognized activities of BPDO[sub B356] include the 2,3-dihydroxyation of 2,4,4'-triCl biphenyl as well as the 2,3- and 3,4- dihydroxyation of 2,6-diCl biphenyl. For BPDO[sub LB400] and BPDO[sub B356], the degree of uncoupling was inversely related to how well the congener was transformed. The PCB-transforming abilities of BPDO₁₁₉ and BPDO₁₁₁₀ were more similar to those of BPDO[sub LB400]. However, both showed improved ability to transform either para- (BPDO₁₁₉) or meta-substituted congeners (BPDO₁₁₁₀). The crystal structures of BPDO[sub LB400] and BPDO₁₁₉ further confirmed the role of residues in region III in the range of substrates accepted by BPDO.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat