Substrate Report for: Epoxyhexane

Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two alpha/beta hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85 degrees C retaining 80% of its initial activity after 3 h thermal treatment at 70 degrees C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 A, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step alpha/beta-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection.

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two alpha/beta hydrolase superfamily fold enzymes were cloned, over-expressed in Escherichia coli, purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of cis-2,3-epoxybutane producing the (2R,3R)-diol product with ee exceeding 99%. Interestingly these enzymes also hydrolyse (4R)-limonene-1,2-epoxide with Sibe-EH being specific for the trans isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85 degrees C retaining 80% of its initial activity after 3 h thermal treatment at 70 degrees C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 A, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step alpha/beta-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection.

The CFTR inhibitory factor (Cif) is an epoxide hydrolase (EH) virulence factor secreted by the bacterium Pseudomonas aeruginosa. Sequence alignments reveal a pattern of Cif-like substitutions that proved to be characteristic of a new subfamily of bacterial EHs. At the same time, crystallographic and mutagenetic data suggest that EH activity is required for virulence and that Cif's active site remains generally compatible with a canonical two-step EH mechanism. A hallmark of this mechanism is the formation of a covalent hydroxyalkyl-enzyme intermediate by nucleophilic attack. In several well-studied EHs, this intermediate has been captured at near stoichiometric levels, presumably reflecting rate-limiting hydrolysis. Here we show by mass spectrometry that only minimal levels of the expected intermediate can be trapped with WT Cif. In contrast, substantial amounts of intermediate are recovered from an active-site mutant (Cif-E153Q) that selectively targets the second, hydrolytic release step. Utilizing Cif-E153Q and a previously reported nucleophile mutant (Cif-D129S), we then captured Cif in the substrate-bound, hydroxyalkyl-intermediate, and product-bound states for 1,2-epoxyhexane, yielding the first crystallographic snapshots of an EH at these key stages along the reaction coordinate. Taken together, our data illuminate the proposed two-step hydrolytic mechanism of a new class of bacterial virulence factor. They also suggest that the failure of WT Cif to accumulate a covalent hydroxyalkyl-enzyme intermediate reflects an active-site chemistry in which hydrolysis is no longer the rate-limiting step, a noncanonical kinetic regime that may explain similar observations with a number of other EHs.

Chlorinated ethenes are the most prevalent ground-water pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation. Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases. Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111). The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wild-type EchA at low concentrations (6.8 microm) and up to 10-fold at high concentrations (540 microm). EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 microm), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 microm). For complete degradation of cis-DCE to chloride ions, the apparent Vmax/Km for the Escherichia coli strain expressing recombinant the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA. The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).