W0175

Structures of a PCB-Degrading CC-Bond Hydrolase Pertaining to Mechanism and Substrate Preference. Jiyuan Ke¹, Shaodong Dai¹, Stephen Y. K. Seah², Cheryl Whiting², Lindsay D. Eltis² & Jeffrey T. Bolin¹, ¹Markey Center for Structural Biology, Dept. of Biological Sciences, Purdue Univ., W Lafayette, IN 47907-2054, USA; ²Depts. of Microbiology and Biochemistry, Univ. British Columbia, Vancouver, BC, V6T 1Z3, Canada.

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs). The failure of the bph pathway to efficiently process PCBs- and PCB metabolites limits strategies for bioremediation. We study mechanisms of BphD catalysis and catalytic failure using structural and biochemical approaches.

Crystal structures of BphD_LB400 (BphD from Burkholderia strain LB400), its S112C mutant, and the S112C:HOPDA complex have been determined at 1.6 Å resolution. BphD_LB400 is a tetramer. Each monomer is divisible into core and lid domains. The active site is located between the domains and includes a catalytic triad, S112-H265-D237, typical of α/β hydrolases. The positions of the backbone amides of G42 and M113 are appropriate for the expected oxyanion binding site.

The mechanism of CC bond hydrolases is a subject of debate. Recent biochemical evidence favors a mechanism that generates a gem-diol intermediate following base-catalyzed attack by water rather than a serine nucleophile mechanism. In the S112C:HOPDA structure, C112 covalently binds HOPDA at the C6 position, forming a complex that resembles a tetrahedral intermediate consistent with serine-nucleophile mechanism.

Comparisons of BphD_LB400 with homologues suggest that polar residues of the substrate-binding pocket are conserved whereas non-polar residues are not. For example, the preferences of BphD and CumD for different substrates is determined by non conserved non-polar residues.

This research was supported by NIH (GM-52381) and NSERC (STP01923182 and OGP0171359), and various agencies that support BioCARs and SBC at the Advanced Photon Source.