Structural studies of BphD LBb400 variants: Insights into the reaction mechanism of MCP hydrolases

Subhangi Ghosh, Purdue University

Abstract

BphDLB400, a C-C bond hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, is a key determinant in the degradation of biphenyl and PCBs. Homologues play a similar role in the degradation of dioxin and other xenobiotic pollutants. BphDLB400 catalyzes the cleavage of the C5-C6 bond of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA). The reaction is believed to proceed via two phases and is known to be dependent on residues Ser112 and His265. In the first phase HOPDA undergoes tautomerization, which facilitates the second phase, hydrolysis of the C5-C6 bond. The hydrolysis may proceed either by nucleophilic attack by an activated water molecule or by the serine. The present study further explores the roles of Ser112 and His265 in the mechanism. For the wild type enzyme, prior studies demonstrated rapid formation of an intermediate with a spectrum red shifted (λmax=492 nm) from that of the substrate (λmax=434 nm). In the BphD LB400 S112A mutant, this intermediate decays extremely slowly and is effectively trapped. In BphDLB400 variants carrying the mutation H265A, this intermediate is not observed. Prior crystal structures of enzyme:HOPDA complexes revealed markedly different conformations of HOPDA for the S112A and S112A/H265A variants. The differences could be linked to the ability of H265 to act as a base or its hydrogen bonding capacity. To resolve the issue, the present study investigates the interaction of HOPDA with BphDLB400 in the H265Q mutant, a variant that preserves the hydrogen bonding while ablating the ability of the residue to function as a base. The 1.9 Å structure of BphDLB400 S112A/H265Q complex revealed a conformation of HOPDA similar to that observed in the S112A/H265A complex. No interaction was observed between Gln265 and the C2-oxygen, indicating H-bonding at residue 265 is not sufficient for HOPDA to adopt the non-planar conformation associated with the red-shifted intermediate. The 1.6 Å structure of the BphDLB400 H265Q complex provides the first direct evidence of a covalent acyl-enzyme intermediate, which would arise only from the serine nucleophile mechanism. A modified mechanism is proposed wherein HOPDA binds as a 2-keto, 6-oxido enolate. Protonation at C5 is followed by nucleophilic attack by Ser112 at C6 forming a tetrahedral intermediate. Collapse of the intermediate releases the first product, the enolate isomer of 2-hydroxy-penta-2,4-dienoate (HPD) leaving an acyl-enzyme. Additionally, the present study confirms the correlation between the red-shifted intermediate generated in the pre-kinetic assays and the non-planar conformation of HOPDA observed in BphDLB400 S112A complex. This was achieved by coincident single crystal absorption spectroscopy and X-ray diffraction experiments performed on the complex of a homologue, DxnB2 RW1 S105A:HOPDA. DxnB2RW1 is the hydrolase in the dibenzofuran degradation pathway, which has the ability to degrade 3-Cl and 4-OH HOPDAs, reported inhibitors of BphD.

Degree

Ph.D.

Advisors

Bolin, Purdue University.

Subject Area

Biochemistry|Biophysics

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