Abstract
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 α/β 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°C retaining 80% of its initial activity after 3 h thermal treatment at 70°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 Å, 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.
Highlights
Epoxide hydrolases (EHs, EC 3.3.2.9) are enzymes that catalyze the in vivo hydrolysis of an epoxide ring to the corresponding vicinal diols
When the metagenomic open reading frames (ORFs) identified in the HotZyme project were aligned with the amino acid sequences of the well characterized α/β epoxide hydrolase (EH) from Agr. radiobacter, B. megaterium, Asp. niger, and S. tuberosum, as well as the metagenome derived enzyme Kau2-EH, two ORFs showed good similarity
It is worth noting that the new EH α/β class homologs were identified in the same two environments, where we had previously isolated the limonene EHs (LEHs) which belong to a different structural family (Ferrandi et al, 2015b)
Summary
Epoxide hydrolases (EHs, EC 3.3.2.9) are enzymes that catalyze the in vivo hydrolysis of an epoxide ring to the corresponding vicinal diols. The physiological roles of EHs have been widely studied These enzymes have been shown to be involved in the detoxification of xenobiotics in mammals (Oesch, 1973; Morisseau and Hammock, 2005) and fungi (Sutherland, 1992), in the general defense system against pathogens and stress response in plants (Kiyosue et al, 1994; Guo et al, 1998) and in hormone biosynthesis in insects (Linderman et al, 2000).
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