Structural investigation into the mechanism of catalysis and inhibition of two carbon -carbon bond hydrolases

Shivarupam Bhowmik, Purdue University

Abstract

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD) is a key enzyme involved in the aerobic transformation of polychlorinated biphenyls (PCBs), a common environmental pollutant. BphD catalyzes an unusual C–C bond hydrolysis, and it has been proposed that His-mediated enol-keto tautomerization precedes cleavage by serine-activated attack of water to form a gem -diol intermediate. Failure of BphD to efficiently process some PCB-metabolites, such as 3-Cl HOPDA, which has an inhibitory effect, limits strategies for bioremediation. Crystal structures of BphDLB400 and its Ser112Ala variant with and without HOPDA and 3-Cl HOPDA were determined to probe the mechanism of catalysis and inhibition. The 1.8 Å resolution structure of BphD LB400-Ser112Ala:HOPDA implicates specific residues in binding and catalysis. Consistent with the analysis of kinetic and spectroscopic data, HOPDA is observed as the keto isomer, with the C2-oxygen atom oriented towards His265 implying its role in tautomerization. Moreover, superposition of the structures of HOPDA complexes onto the wild type structure suggests a mechanism of hydrolysis consistent with a serine-nucleophile mechanism, as solvent molecules have poor access to the scissile bond. The 1.7 Å resolution structure of BphDLB400-Ser112Ala:3-Cl HOPDA revealed 3-Cl HOPDA bound as an enol isomer in a non-productive binding mode. Binding of the C3-Cl atom in a hydrophobic pocket is a plausible cause of the non-productive binding mode and catalytic impairment. This roadblock to PCB bioremediation might be overcome by exploiting the complementary substrate specificity of a homologue, DxnB2RW1. The enzyme processes 3-Cl HOPDA with a ∼28-fold higher catalytic rate constant (kcat). The crystal structure of DxnB2 RW1 reveals a similar monomeric structure to that of BphDLB400 , with the active site located at a cleft between two domains: a core domain and a helical lid domain. The 2.0 Å resolution crystal structure of the complex of 3-Cl HOPDA with the catalytically impaired Ser105Ala variant suggests weaker interaction of C3-Cl atom in a hydrophobic pocket in the active site may allow 3-Cl HOPDA to assume a productive conformation. Moreover, a transition to a productive binding mode may also be facilitated by structural flexibility of the lid domain.

Degree

Ph.D.

Advisors

Bolin, Purdue University.

Subject Area

Biochemistry|Biophysics

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