Recalcitrant environmental pollutants, like bromoorganics and epoxides are hydrolysed with limited substrate specificities by microbial oxygenases, reductases, hydrolases and dehalogenases. Here, we report the identification and characterisation of a protein (XP_504164) from the tropical marine yeast Yarrowia lipolytica NCIM 3589, known to degrade bromoorganics and epoxides. Multiple sequence alignment suggests it belongs to alpha/beta superfamily with conservation of catalytic triad and oxyanion hole motifs. The corresponding gene cloned and protein (Ylehd) expressed in E. coli BL21AI exhibited epoxide hydrolase activity (24 +/- 0.7 nmol s-1 mg-1 protein) at pH 8.0 and promiscuous haloalkane dehalogenase (1.5 +/- 0.2 nmol s-1 mg-1 protein) at pH 4.5. Recombinant Ylehd catalyses structurally diverse epoxides and bromoorganics with maximum catalytic efficiency (kcat/Km) of 96.56 and 10.1 mM-1 s-1 towards 1,2-Epoxyoctane (EO) and 1-Bromodecane (BD). The expression of Ylehd was highly induced in presence of BD and EO but not in glucose grown cells as studied by immunoblot analyses, q-PCR and activity levels. Immunoelectron microscopy confirmed higher expression in presence of xenobiotics and located it to cytosol. Such inducible nature of Ylehd suggests its physiological role in xenobiotic stress mitigation. This study represents the first functional characterisation of a bifunctional EH/HLD in eukaryotic microbes with broad substrate specificity making it a potential biocatalyst for bioremediation/biosensing of mixed pollutants.
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.
Title: Cloning and characterization of an epoxide hydrolase from Cupriavidus metallidurans-CH34 Kumar R, Wani SI, Chauhan NS, Sharma R, Sareen D Ref: Protein Expr Purif, 79:49, 2011 : PubMed
A putative epoxide hydrolase-encoding gene was identified from the genome sequence of Cupriavidus metallidurans CH34. The gene was cloned and overexpressed in Escherichia coli with His(6)-tag at its N-terminus. The epoxide hydrolase (CMEH) was purified to near homogeneity and was found to be a homodimer, with subunit molecular weight of 36 kDa. The CMEH had broad substrate specificity as it could hydrolyze 13 epoxides, out of 15 substrates tested. CMEH had high specific activity with 1,2-epoxyoctane, 1,2-epoxyhexane, styrene oxide (SO) and was also found to be active with meso-epoxides. The enzyme had optimum pH and temperature of 7.5 and 37 degrees C respectively, with racemic SO. Biotransformation of 80 mM SO with recombinant whole E. coli cells expressing CMEH led to 56% ee(P) of (R)-diol with 77.23% conversion in 30 min. The enzyme could hydrolyze (R)-SO, approximately 2-fold faster than (S)-SO, though it accepted both (R)- and (S)-SO with similar affinity as K(m)(R) and K(m)(S) of CMEH were 2.05+/-0.42 and 2.11+/-0.16 mM, respectively. However, the k(cat)(R) and k(cat)(S) for the two enantiomers of SO were 4.80 and 3.34 s(-1), respectively. The wide substrate spectrum exhibited by CMEH combined with the fast conversion rate makes it a robust biocatalyst for industrial use. Regioselectivity studies with enantiopure (R)- and (S)-SO revealed that with slightly altered regioselectivity, CMEH has a high potential to synthesize an enantiopure (R)-PED, through an enantioconvergent hydrolytic process.
