Recombinant 2-enoyl-CoA hydratase derived from rat peroxisomal multifunctional enzyme 2: role of the hydratase reaction in bile acid synthesis

Abstract

Rat liver peroxisomes contain two multifunctional enzymes: (1) perMFE-1 [2-enoyl-CoA hydratase 1/Delta3,Delta2-enoyl-CoA isomerase/(S)-3-hydroxyacyl-CoA dehydrogenase] and (2) perMFE-2 [2-enoyl-CoA hydratase 2/(R)-3-hydroxyacyl-CoA dehydrogenase]. To investigate the role of the hydratase activity of perMFE-2 in beta-oxidation, a truncated version of perMFE-2 was expressed in Escherichia coli as a recombinant protein. The protein catalyses the hydration of straight-chain (2E)-enoyl-CoAs to (3R)-hydroxyacyl-CoAs, but it is devoid of hydratase 1 [(2E)-enoyl-CoA to (3S)-hydroxyacyl-CoA] and (3R)-hydroxyacyl-CoA dehydrogenase activities. The purified enzyme (46 kDa hydratase 2) can be stored as an active enzyme for at least half a year. The recombinant enzyme hydrates (24E)-3alpha,7alpha,12alpha-trihydroxy- 5beta-cholest-24-enoyl-CoA to (24R,25R)-3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholestanoyl-CoA, which has previously been characterized as a physiological intermediate in bile acid synthesis. The stereochemistry of the products indicates that the hydration reaction catalysed by the enzyme proceeds via a syn mechanism. A monofunctional 2-enoyl-CoA hydratase 2 has not been observed as a wild-type protein. The recombinant 46 kDa hydratase 2 described here survives in a purified form under storage, thus being the first protein of this type amenable to application as a tool in metabolic studies.

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

“…Samples were further prepared according to the instructions for electrophoresis of NuPAGE Bis-Tris mini gels Hsd17b4 seems to be able to handle all peroxisomal substrates ( 7 ), which is in line with earlier biochemical studies using purifi ed enzymes (8)(9)(10)(11)(12). Indeed, HSD17B4-deficient patients and Hsd17b4 KO mice accumulate very long-chain as well as branched-chain fatty acids and bile acid intermediates ( 13,14 ).…”

A Two-domain Structure of One Subunit Explains Unique Features of Eukaryotic Hydratase 2

“…It has been shown by patient mutations as well as in vitro studies that hydratase 2 is a crucial enzyme in lipid metabolism of ␣-methyl-branched fatty acids (e.g. pristanic acid), bile acid intermediates, and very long-chain fatty acids (5)(6)(7)(8).…”

Human Peroxisomal Multifunctional Enzyme Type 2

“…The MFE-2-dependent pathway operates in yeast peroxisomes (3), whereas the MFE-1-dependent pathway, which utilizes (3S)-hydroxyacyl-CoA intermediates, like mitochondrial and bacterial ␤-oxidation, is also found in mammalian peroxisomes (6 -8). The MFE-2 polypeptide has been found by amino acid sequence alignment to have three putative functional domains, and its modular organization has been verified experimentally (9,10). The data show that the NH 2 -terminal domain, which belongs to the short chain alcohol dehydrogenase/reductase superfamily, is responsible for (3R)-hydroxyacyl-CoA dehydrogenase activity.…”

“…Construction of the Plasmid pET3a::HsMFE-2(dh⌬)-A cDNA fragment encoding residues 319 -735 of HsMFE-2 (23) was chosen because the corresponding fragment from the rat had been shown to encode a stable protein (10). The fragment was amplified by polymerase chain reaction with HsMFE-2-specific primers: 5Ј-primer, catatgACA GCA ACA TCA GGA TTT GCT G and 3Ј-primer, ggatccTCA GAG CTT GGC GTA GTC TTT AAG.…”