title: Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme.
abstract: In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involved in fatty acid and cholesterol side-chain beta-oxidation. Mammalian peroxisomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. As has been shown previously, perMFE-2 hydrates (24E)-3alpha,7alpha, 12alpha-trihydroxy-5beta-cholest-24-enoyl-CoA to (24R, 25R)-3alpha, 7alpha,12alpha,24xi-tetrahydroxy-5beta-choles tanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3alpha,7alpha, 12alpha,24xi-tetrahydroxy-5beta-cholestanoyl-CoA , the mammalian perMFE-2 dehydrogenates only the (24R,25R)-isomer. The yeast peroxisomal multifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R)-3-hydroxyacyl-CoA dehydrogenase. To investigate the stereospecificity of yeast peroxisomal MFE, the two dehydrogenase domains of C. tropicalis MFE were expressed in E. coli as a 65 kDa recombinant protein. This protein catalyzes the dehydrogenation of straight-chain (R)-3-hydroxyacyl-CoAs, but it is devoid of (S)-3-hydroxyacyl-CoA dehydrogenase and 2-enoyl-CoA hydratase activities. The protein dehydrogenates (24R,25R)- and (24R, 25S)-isomers of 3alpha,7alpha, 12alpha, 24xi-tetrahydroxy-5beta-cholestanoyl-CoA. Interestingly, the protein also shows 17beta-estradiol dehydrogenase activity. As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemistry of bile acid synthesis.
authors.affiliation: Biocenter Oulu, University of Oulu, FIN-90570 Oulu, Finland.
authors.forename: Y M
authors.initials: YM
authors.lastname: Qin
authors.forename: M H
authors.initials: MH
authors.lastname: Poutanen
authors.forename: D K
authors.initials: DK
authors.lastname: Novikov
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002175:Candida / Q000201:enzymology*; D017931:DNA Primers; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020675:Peroxisomes / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D015094:3-Hydroxyacyl CoA Dehydrogenases; D000426:Alcohol Dehydrogenase