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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)-crotonyi-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 C[O.sub.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. alcohol dehydrogenase | biocatalysis | enoyl reductase

Ruminal administration of a triple indigestible marker system comprised of cobalt EDTA (CoEDTA), ytterbium acetate (YbAc), and chromium-mordanted straw (CrS) decreases product:substrate ratios for [DELTA]9-desaturase in bovine milk fat. This experiment was designed to identify the marker(s) responsible and develop an alternative system for simultaneous determination of nutrient flow in the gastro-intestinal tract and milk fatty acid composition. Five lactating dairy cows were used in a 5 x 5 Latin square with 21-d periods to evaluate the effects of YbAc, CoEDTA, and CrS independently or as part of a triple marker system (TMS), and CrEDTA as an alternative to CoEDTA on milk fat composition. Markers were administered in the rumen over a 7-d interval and samples of milk were collected on d -1, 3, 7, and 11. Both TMS and CoEDTA alone reduced the concentrations of milk fatty acids containing a cis-9 double bond, whereas YbAc, CrS, and CrEDTA had no effect. Reductions in product:substrate ratios for [DELTA]9-desaturase were time dependent and evident within 3 d of administration. Ruminal infusion of CoEDTA for 7 d induced mean decreases in milk cis-9 14:1/14:0, cis-9 16:1/16:0, cis-9 18:1/18:0, and cis-9, trans-11 conjugated linoleic acid/trans-11 18:1 concentration ratios of 47.7, 26.7, 40.3, and 42.6%, respectively. In conclusion, ruminal infusion of CoEDTA alters milk fatty acid composition and appears to inhibit [DELTA]9-desaturase activity in the bovine mammary gland. Results indicate that a TMS based on CrEDTA, YbAc, and indigestible neutral detergent fiber can be used for estimating nutrient flow without altering milk fat composition in lactating cows.

The mechanism of temperature adaptation in plants, including the formation of polyunsaturates in seed storage lipids, most likely involves transcriptional as well as post-translational regulation of fatty acid desaturase activity. The present investigation was conducted to measure changes in the transcript accumulation among the three members of the soybean [Glycine max (L.) Merr.] microsomal omega-3 fatty acid desaturase gene family in response to altered growth temperature during seed development. Microsomal omega-3 fatty acid desaturases catalyze the insertion of a third double bond into linoleic ([18:2.sup.[DELTA]9], 12) acid to produce linolenic ([18:3.sup.[DELTA]9], 12,15) acid. At 35 d after flowering, transcript accumulation (normalized for soybean actin) of GmFAD3A decreased by 5- to 15-fold, GmFAD3B by 2- to 9-fold, and GmFAD3C by 2- to 3-fold in seeds that developed in a warm (day/ night [D/N] = 30/26[degrees]C) versus a normal (D/N = 26/22[degrees]C) or a cool (D/N = 22/18[degrees]C) environment. At this stage of seed development, decreased omega-3 desaturase gene expression levels were positively associated with reductions of 39 to 50% in the linolenic acid content of seeds of three soybean varieties examined. Thus, transcriptional regulation of the microsomal omega-3 fatty acid desaturase gene family likely accounts, at least in part, for the reduced linolenic acid levels in soybean seeds grown at elevated temperature.

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the first step of lipid A biosynthesis, the reversible transfer of the R-3-hydroxyacyl chain from R-3-hydroxyacyl acyl carrier protein to the glucosamine 3-OH group of UDP-GlcNAc. Escherichia coli LpxA is highly selective for R-3-hydroxymyristate. The crystal structure of the E. coli LpxA homotrimer, determined previously in the absence of lipid substrates or products, revealed that LpxA contains an unusual, left-handed parallel [beta]-helix fold. We have now solved the crystal structures of E. coli LpxA with the bound product UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc at a resolution of 1.74 [Anstrom] and with bound UDP-3-O-(R-3-hydroxydecanoyl)GlcNAc at 1.85 [Angstrom]. The structures of these complexes are consistent with the catalytic mechanism deduced by mutagenesis and with a recent 3.0-[Angtrom] structure of LpxA with bound UDP-GlcNAc. Our structures show how LpxA selects for 14-carbon R-3-hydroxyacyl chains and reveal two modes of UDP binding. UDP-3-O-(R-3-hydroxymyristoyl)-acetylglucosamine | acyl carrier protein LpxA | Escherichia coli | x-ray crystallography

Peroxisome proliferator-activated receptor alpha (PPARalpha) binds with high affinities the very-long-chain acyl CoAs (VLCFA-CoAs), and with slightly weaker affinities the free branched-chain fatty acids (BCFA). The high affinity binding of the ligand correlate with ligand-induced conformational changes in PPARalpha, eliciting alterations in coactivator recruitment.

Stearoyl-acyl carrier protein desaturase (delta9D) catalyzes the O(sub 2) and 2e(super -) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield obeoyl-ACP (18:1-ACP). These protein-protein interface involved in the Fd-delta9D complex is investigated by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies.

Potential of mean force calculations were performed on the wild-type medium-chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. The study demonstrates that significant insights into the catalytic mechanism can be obtained from simulation studies, and it is possible to design novel mechanistic probes for the enzyme with the help of the result.

The thermodynamic regulation is examined through direct characterization of the electrochemical properties of human short-chain acyl-CoA dehydrogenase (hSCAD) using spectroelectrochemical methodology. The result indicates that in hSCAD, substrate redox activation occurs that leads to a large enzyme midpoint potential shift and substrate binding appear to make a larger contribution than does product to thermodynamic modulation.

Short-chain acyl-CoA dehydrogenase (hSCAD) catalyzes the first matrix step in the mitochondrial beta-oxidation cycle with optimal activity toward butyryl- and hexanoyl-CoA. The result provides valuable insight into the mechanistic basis for dysfunction of the common variant hSCADs and demonstrates that mutations, regardless of their position in the protein structure, have a large impact on the redox properties of the enzyme.

The cloning and characterization of the oleate desaturase from basidiomycete Phanerochaete chrysosporium which contains a single integral membrane [[Delta].sup.12]-desaturase FAD2 related to the endoplasmic reticular plant FAD2 enzymes is reported. The results provide insight into the activity of the fatty acyl [[Delta].sup.12]-desaturase family and the evolution of basidiomycete fad2 genes from an ancestral desaturase.

PksE is the first polyunsaturated fatty acid (PUFA)-like polyketide enzyme that is identified as a trans-acting enoyl reductase (ER). The results have disclosed a general enoyl reduction pathway in polyketide biosynthesis and have described a strategy by which trans-enoyl reductases are used for modulating small-molecule function.

The triacylglycerol (TAG) biosynthesis in Streptomyces coelicolor is studied and the roles of the three putative wax ester synthase (WS)/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (DGAT) proteins Sco0958, Sco0123 and Sco1280 in the accumulation of neutral lipids are evaluated. It is shown that the residual production of TAG in this mutant strain is mediated by an acyl-CoA-dependent pathway, as the triple mutant has displayed DGAT activity.

The remarkable substrate tolerance of the fatty acyl-CoA ligases (FACLs) to produce diverse acyl-CoAs and N-acetylcysteamine (NAC) analogues from their corresponding acids is reported. The acyl-activating enzymes (AAE) proteins provide an attractive route to expand the precursors available for polyketide biosynthesis.

Model systems for the reactant complex of tricyclic or bicyclic models of FAD (Fl (sub OX)) with the thioenolate portion of acyl-CoA are investigated to study the role of charge transfer (CT) interactions in the catalytic activity of acyl-CoA dehydrogenase. Analysis of the charge distribution in thioenolate/Fl (sub OX) complexes indicates significant charge transfer to flavin (about 0.7 e).