The long form of human uncoupling protein-3 (hUCP3L) is highly homologous to thermogenin (UCP1), the uncoupling protein of brown fat mitochondria, but is expressed predominantly in skeletal muscle. Its putative role is to regulate the coupling efficiency of oxidative phosphorylation and thus thermogenesis in skeletal muscle, a major thermogenic tissue in higher mammals. To study the functional relevance of hUCP3L, the protein was expressed in yeast cells under the control of the galactose promoter. Expression of hUCP3L induced a series of phenotype changes in the yeast cells. The cellular growth and the mitochondrial membrane potential were both diminished. The portion of cellular respiration coupled to oxidative phosphorylation decreased from 57% to 11% (P 6 0.001) and the cellular heat production, as measured by direct microcalorimetry, was increased by 33.3 þ 3.2% (P 6 0.001) after induction of UCP3L. These observations demonstrate for the first time the intrinsic thermogenic properties of hUCP3L in intact cells.z 1999 Federation of European Biochemical Societies.

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of n/~-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with 11 -"C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1 ,I ,I -trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalconeoxide-senaitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-'JC]cholesterol-5a,6n-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.Keywords ; epoxide hydrolase ; mechanism ; a/b hydrolase fold ; cholesterol ; fatty acid metabolism.Epoxide hydrolases (EH) represent a group of ubiquitous enzymes with important functions in the detoxification of reactive intermediates, namely epoxides, that arise from a large variety of compounds during their metabolism. The two mammalian enzymes implicated in the metabolism of foreign compounds are microsomal EH (Oesch, 1973) and soluble EH (Ota and Hammock, 1980). Both enzymes have been cloned from a variety of species (Beetham et al., 1993;Grant et al., 1993;Jackson et al., 1987;Knehr et al., 1993;Porter et al., 1986;Wojtasek and Prestwich, 1996). A third enzyme, cholesterol EH, which is membrane bound similarly to inicrosomal EH but otherwise distinct from the latter (Oesch et al., 1984), is less well investigated. Its physiological function appears to be the conversion of 5n,6n-epoxycholestane-3P-o1 and SP,6D-epoxycholestane-3/!-ol, the two epoxides arising from cholesterol during, e.g. lipid peroxidation, to give the single product cholestane-3~,Scr,b/j-triol (Watabe et al., 1981).For a long time it was believed that EH convert their substrates by direct hydrolysis. The pioneering work of Hanzli...

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

Two splice variants of the human uncoupling protein-3 (UCP3L and UCP3S) are highly expressed in skeletal muscle. The properties of UCP3L and S have been compared to those of UCP1 in a heterologous yeast expression system under the control of the galactose promoter. Both UCP3 isoforms were found to strongly impair the coupling efficiency of respiring cells thus resulting in increased thermogenesis. The uncoupling properties of both UCP3L and S could be clearly demonstrated also in isolated yeast mitochondria both in terms of coupled respiration and in the capacity to polarize the inner membrane in conditions of limited substrate availability. Contrary to what was observed with mitochondria containing UCP1, millimolar GDP and ATP had little if any effect on the uncoupling activity of UCP3. A very marked uncoupling of whole cells and isolated mitochondria was observed at very low expression levels of UCP3S indicating that the short isoform is more active than the long one.z 1999 Federation of European Biochemical Societies.

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.