Steady-state kinetic characterization and crystallization of a polychlorinated biphenyl-transforming dioxygenase.
The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe(2)S(2) center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 A. Steady-state kinetics assays demonstrated that BPDO had an apparent k(cat)/K(m) for biphenyl of (1.2 +/- 0.1) x 10(6) M(-1) s(-1) in air-saturated buffer. Moreover, BPDO transformed dichlorobiphenyls (diClBs) in the following order of apparent specificities: 3,3'- > 2,2'- > 4, 4'-diClB. Strikingly, the ability of BPDO to utilize O(2) depended strongly on the biphenyl substrate: k(cat)/K(m(O(2))) = (3.6 +/- 0. 3), (0.06 +/- 0.02), and (0.4 +/- 0.07) x 10(5) M(-1) s(-1) in the presence of biphenyl and 2,2'- and 3,3'-diClBs, respectively. Moreover, biphenyl/O(2) consumed was 0.97, 0.44, 0.63, and 0.48 in the presence of biphenyl and 2,2'-, 3,3'-, and 4,4'-diClBs, respectively. Within experimental error, the balance of consumed O(2) was detected as H(2)O(2). Thus, PCB congeners such as 2, 2'-diClB exact a high energetic cost, produce a cytotoxic compound (H(2)O(2)), and can inhibit degradation of other congeners. Each of these effects would be predicted to inhibit the aerobic microbial catabolism of PCBs.
Imbeault
N Y
NY
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec H3G 1M8, Canada.
Powlowski
J B
JB
Colbert
C L
CL
Bolin
J T
JT
Eltis
L D
LD
D049308:Dioxygenases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011078:Polychlorinated Biphenyls / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014961:X-Ray Diffraction
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D011078:Polychlorinated Biphenyls; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase; C074161:biphenyl-2,3-dioxygenase; D010100:Oxygen