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UBC Theses and Dissertations

Characterization of BphD, a C-C bond hydrolase involved in the degradation of polychlorinated biphenyls Horsman, Geoffrey

Abstract

Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme.

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