Publications

• Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase.

  Erb T.J., Brecht V., Fuchs G., Mueller M., Alber B.E.

  Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which cataly ... >> More

  Chemo- and stereoselective reductions are important reactions in chemistry and biology, and reductases from biological sources are increasingly applied in organic synthesis. In contrast, carboxylases are used only sporadically. We recently described crotonyl-CoA carboxylase/reductase, which catalyzes the reduction of (E)-crotonyl-CoA to butyryl-CoA but also the reductive carboxylation of (E)-crotonyl-CoA to ethylmalonyl-CoA. In this study, the complete stereochemical course of both reactions was investigated in detail. The pro-(4R) hydrogen of NADPH is transferred in both reactions to the re face of the C3 position of crotonyl-CoA. In the course of the carboxylation reaction, carbon dioxide is incorporated in anti fashion at the C2 atom of crotonyl-CoA. For the reduction reaction that yields butyryl-CoA, a solvent proton is added in anti fashion instead of the CO(2). Amino acid sequence analysis showed that crotonyl-CoA carboxylase/reductase is a member of the medium-chain dehydrogenase/reductase superfamily and shares the same phylogenetic origin. The stereospecificity of the hydride transfer from NAD(P)H within this superfamily is highly conserved, although the substrates and reduction reactions catalyzed by its individual representatives differ quite considerably. Our findings led to a reassessment of the stereospecificity of enoyl(-thioester) reductases and related enzymes with respect to their amino acid sequence, revealing a general pattern of stereospecificity that allows the prediction of the stereochemistry of the hydride transfer for enoyl reductases of unknown specificity. Further considerations on the reaction mechanism indicated that crotonyl-CoA carboxylase/reductase may have evolved from enoyl-CoA reductases. This may be useful for protein engineering of enoyl reductases and their application in biocatalysis. << Less

  Proc. Natl. Acad. Sci. U.S.A. 106:8871-8876(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural enzymological studies of 2-enoyl thioester reductase of the human mitochondrial FAS II pathway: new insights into its substrate recognition properties.

  Chen Z.J., Pudas R., Sharma S., Smart O.S., Juffer A.H., Hiltunen J.K., Wierenga R.K., Haapalainen A.M.

  Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the bi ... >> More

  Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway. << Less

  J. Mol. Biol. 379:830-844(2008) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli.

  Wallace K.K., Bao Z.Y., Dai H., Digate R., Schuler G., Speedie M.K., Reynolds K.A.

  A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for croton ... >> More

  A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for crotonyl-CoA and 15 microM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki = 9.5 microM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki > 400 microM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein. << Less

  Eur. J. Biochem. 233:954-962(1995) [PubMed] [EuropePMC]