The Molecular Structure of Epoxide Hydrolase B from Mycobacterium tuberculosis and Its Complex with a Urea-Based Inhibitor

Abstract

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC 50 ≈19 nM) urea-based inhibitor at 2.1 and 2.4 Å resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical α/β hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the bindingsite residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120° about its C α -C β bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the α/β type. KeywordsMycobacterium tuberculosis; epoxide hydrolase; hydrolase fold; enzyme mechanism; inhibitor design *Corresponding author. Michael.James@ualberta.ca. Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jmb.2008.06.030 Protein Data Bank accession codesThe atomic coordinates and the structure factors for the free Mtb EHB and Mtb EHB bound to the inhibitor structures have been deposited in the Protein Data Bank|| with the accession codes 2E3J and 2ZJF, respectively.

Gates of Enzymes

“…67 The cap domain movement of epoxide hydrolase from Mycobacterium tuberculosis is another enzyme with a domain-scale gate that controls substrate access to the active site cavity. 68 Monomers of phospholipase A2 control access to their interface and the active site by adopting a different conformation during dimer aggregation. 69 Large domain movements often require an additional source of energy.…”

Single-Nucleotide Polymorphisms in PigEPHX1Gene are Associated with Pork Quality Traits

“…The structure of epoxide hydrolase B consists of a monomer that maintains alpha=beta hydrolase fold that consists of catalytic domain and cap domain that regulates the active site for hydrolysis. (5). The amino acid residues of Asp104, His333, and Asp302 in EPHX1 form a catalytic triad that plays a critical enzymatic role.…”

Exported Epoxide Hydrolases Modulate Erythrocyte Vasoactive Lipids during Plasmodium falciparum Infection

“…In the assays reported here, PfEH1 and PfEH2 behaved similarly (the exception being that PfEH2 did not hydrolyze the EF7 fluorescent reporter substrate, though EHs with poor activity against nonphysiological fluorescent reporters but higher activity against endogenous substrates have been reported [59]). If the localization and ability to hydrolyze EETs/EpOMEs are similar, then it is unclear why P. falciparum would express two EHs.…”