Cloning and molecular characterization of a soluble epoxide hydrolase from Aspergillus niger that is related to mammalian microsomal epoxide hydrolase

Abstract

Aspergillus niger strain LCP521 harbours a highly processive epoxide hydrolase (EH) that is of particular interest for the enantioselective bio-organic synthesis of fine chemicals. In the present work, we report the isolation of the gene and cDNA for this EH by use of inverse PCR. The gene is composed of nine exons, the first of which is apparently non-coding. The deduced protein of the A. niger EH shares significant sequence similarity with the mammalian microsomal EHs (mEH). In contrast to these, however, the protein from A. niger lacks the common N-terminal membrane anchor, in line with the fact that this enzyme is, indeed, soluble in its native environment. Recombinant expression of the isolated cDNA in Escherichia coli yielded a fully active EH with similar characteristics to the fungal enzyme. Sequence comparison with mammalian EHs suggested that Asp(192), Asp(348) and His(374) constituted the catalytic triad of the fungal EH. This was subsequently substantiated by the analysis of respective mutants constructed by site-directed mutagenesis. The presence of an aspartic acid residue in the charge-relay system of the A. niger enzyme, in contrast to a glutamic acid residue in the respective position of all mEHs analysed to date, may be one important contributor to the exceptionally high turnover number of the fungal enzyme when compared with its mammalian relatives. Recombinant expression of the enzyme in E. coli offers a versatile tool for the bio-organic chemist for the chiral synthesis of a variety of fine chemicals.

Soybean Epoxide Hydrolase

“…step of the enzymatic reaction (39). We found that in soybean EH, substitution of Asp 285 by a glutamate affected only moderately its V max , which decreased from 18.0 (wild-type) to 3.75 mol/min/mg, indicating that this location of the active site can accommodate, without too great a consequence, another acidic residue.…”

“…We speculated that if conversely, in soybean EH, the breakdown of the intermediate of the enzyme is more rapid than the alkylation step (k 2 in Equation 2), this change in the rate-limiting step might hinder the accumulation of E-I and make it more difficult to evidence. This would not be without precedent because a fungal EH was also suggested to have a slow alkylation step (39).…”

Biochemical Evidence for the Involvement of Tyrosine in Epoxide Activation during the Catalytic Cycle of Epoxide Hydrolase

“…These enzymes are widely distributed among many species, including bacteria, fungi, plants, insects, and mammals (2)(3)(4)(5). In mammals, there are two major classes of EH with broad and complementary substrate selectivity, soluble EH (sEH) and microsomal EH (mEH) (6).…”

Epoxide Hydrolase‐Catalysed Kinetic Resolution of a Spiroepoxide, a Key Building Block of Various 11‐Heterosteroids

“…[3] As far as spiroepoxides are concerned, if the kinetic resolution of a range of methyl-substituted 1-oxaspiro-A C H T U N G T R E N N U N G [2,5]octanes by the yeast epoxide hydrolase from Rhodotorula glutinis has been recently described, [4] no example of resolution of 1-oxaspiroA C H T U N G T R E N N U N G [2,4]heptanes has been ever carried out. To perform the kinetic resolution of rac-1 we tested two very different EHs, a fungal and a bacterial EH.…”