W0252

Anaerobic Crystal Structures of Biphenyl Dioxygenase and its Complexes With Substrate and Product. Pravindra Kumar¹, Christopher L. Colbert¹, Nathalie Y. R. Agar², Lindsay D. Eltis³, Justin Powloski², and Jeffrey T. Bolin¹, ¹Markey Center for Structural Biology, Dept. of Biological Sciences, Purdue Univ., W Lafayette, IN 47907, USA; ²Dept. of Chemistry and Biochemistry, Concordia Univ., Montreal, PQ, H3G 1M8, Canada; ³Depts. of Microbiology and Biochemistry, Univ. British Columbia, Vancouver, BC, V6T 1Z3, Canada.

Improvements in purification and crystallization led to determination of crystal structures for biphenyl dioxygenase, BPDO, from Comamonas testosteroni strain B-356 and its complexes with substrate and product at 1.5-1.6Å resolution in the presence of full complements of the native metals. Comparisons of free enzyme with the complexes provide evidence that the coordination of the Fe(II) site changes with the state of the enzyme. The internal rearrangements of ligands may be required to avoid steric clashes with substrate and product, may also be important for control of dioxygen binding and activation, and may influence substrate preferences.

Biphenyl dioxygenase is the catalytic component from the multicompon-ent biphenyl dioxygenase system. The other components include a Rieske ferredoxin, which supplies electrons to BPDO, and a flavoprotein ferredoxin reductase. This system catalyzes the first step in the aerobic microbial degradation of biphenyl and some PCBs, and is targeted for improvements to facilitate the bioremediation of PCBs. The chemistry typically involves the dihydroxylation of an aromatic ring to generate a substituted cis-cyclohexadienediol. BPDO is an α₃β₃ hexamer (225 kDa) that binds one Rieske Fe-S cluster and one Fe(II) ion per α subunit. Prior crystallographic studies of BPDO were complicated by loss of Fe(II) from the active site.