Substrate specificities of peroxisomal members of short-chain alcohol dehydrogenase superfamily: expression and characterization of dehydrogenase part of Candida tropicalis multifunctional enzyme

In addition to several other enzymes, the short-chain alcohol dehydrogenase superfamily includes a group of peroxisomal multifunctional enzymes involv ed in fatty acid and cholesterol side-chain beta-oxidation, Mammalian perox isomal multifunctional enzyme type 2 (perMFE-2) is a 2-enoyl-CoA hydratase- 2/(R)-3-hydroxyacyl-CoA dehydrogenase, As has been shown previously, perMFE -9 hydrates (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-eno yl-CoA to (24R, 25R)-3 alpha,7 alpha,12 alpha,24 xi-tetrahydroxy-5 beta-cho lestanoyl-CoA, which has been characterized as a physiological intermediate in cholic acid synthesis. Out of four possible stereoisomers of 3 alpha,7 alpha,12 alpha,24 xi-tetrahydroxy-5 beta-cholestanoyl-CoA, the mammalian pe rMFE-2 dehydrogenates only the (24R,25R)-isomer, The yeast peroxisomal mult ifunctional enzyme (MFE) was first described as 2-enoyl-CoA hydratase-2/(R) -3-hydroxyacyl-CoA dehydrogenase, To investigate the stereospecificity of y east peroxisomal RIFE, the two dehydrogenase domains of C. tropicalis MFE w ere expressed in E. coil as a 65 kDa recombinant protein, This protein cata lyzes 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 3 alpha,7 alpha,12 alpha,24 xi-tehahydrox-5 beta-cholestanoyl-CoA. Interest ingly, the protein also shows 17 beta-estradiol dehydrogenase activity. As a monofunctional (R)-specific 3-hydroxyacyl-CoA dehydrogenase is currently unavailable, this recombinant enzyme can be used to study the stereochemist ry of bile acid synthesis.