Structural enzymology studies with the substrate 3S-hydroxybutanoyl-CoA: bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD.

Multifunctional enzyme, type-1 (MFE1) catalyzes the second and third step of the ß-oxidation cycle, being, respectively, the 2E-enoyl-CoA hydratase (ECH) reaction (N-terminal part, crotonase fold) and the NAD+-dependent, 3S-hydroxyacyl-CoA dehydrogenase (HAD) reaction (C-terminal part, HAD fold). Structural enzymological properties of rat MFE1 (RnMFE1) as well as of two of its variants, namely the E123A variant (a glutamate of the ECH active site is mutated into alanine) and the BCDE variant (without domain A of the ECH part), were studied, using as substrate 3S-hydroxybutanoyl-CoA. Protein crystallographic binding studies show the hydrogen bond interactions of 3S-hydroxybutanoyl-CoA as well as of its 3-keto, oxidized form, acetoacetyl-CoA, with the catalytic glutamates in the ECH active site. Pre-steady state binding experiments with NAD+ and NADH show that the kon and koff rate constants of the HAD active site of monomeric RnMFE1 and the homologous human, dimeric 3S-hydroxyacyl-CoA dehydrogenase (HsHAD) for NAD+ and NADH are very similar, being the same as those observed for the E123A and BCDE variants. However, steady state and pre-steady state kinetic data concerning the HAD-catalyzed dehydrogenation reaction of the substrate 3S-hydroxybutanoyl-CoA show that, respectively, the kcat and kchem rate constants for conversion into acetoacetyl-CoA by RnMFE1 (and its two variants) are about 10 fold lower as when catalyzed by HsHAD. The dynamical properties of dehydrogenases are known to be important for their catalytic efficiency, and it is discussed that the greater complexity of the RnMFE1 fold correlates with the observation that RnMFE1 is a slower dehydrogenase than HsHAD.

Enzymes of the crotonase superfamily: Diverse assembly and diverse function.

The crotonase fold is generated by a framework of four repeats of a ßßα-unit, extended by two helical regions. The active site of crotonase superfamily (CS) enzymes is located at the N-terminal end of the helix of the third repeat, typically being covered by a C-terminal helix. A major subset of CS-enzymes catalyzes acyl-CoA-dependent reactions, allowing for a diverse range of acyl-tail modifications. Most of these enzymes occur as trimers or hexamers (dimers of trimers), but monomeric forms are also observed. A common feature of the active sites of CS-enzymes is an oxyanion hole, formed by two peptide-NH hydrogen bond donors, which stabilises the negatively charged thioester oxygen atom of the reaction intermediate. Structural properties and possible use of these enzymes for biotechnological applications are discussed.

Structural basis for different membrane-binding properties of E. coli anaerobic and human mitochondrial ß-oxidation trifunctional enzymes.

Facultative anaerobic bacteria such as Escherichia coli have two α2ß2 heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the ß-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-ß as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-ß is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-ß dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities.

Engineering a Feruloyl-Coenzyme A Synthase for Bioconversion of Phenylpropanoid Acids into High-Value Aromatic Aldehydes.

Aromatic aldehydes find extensive applications in food, perfume, pharmaceutical, and chemical industries. However, a limited natural enzyme selectivity has become the bottleneck of bioconversion of aromatic aldehydes from natural phenylpropanoid acids. Here, based on the original structure of feruloyl-coenzyme A (CoA) synthetase (FCS) from Streptomyces sp. V-1, we engineered five substrate-binding domains to match specific phenylpropanoid acids. FcsCIAE407A/K483L, FcsMAE407R/I481R/K483R, FcsHAE407K/I481K/K483I, FcsCAE407R/I481R/K483T, and FcsFAE407R/I481K/K483R showed 9.96-, 10.58-, 4.25-, 6.49-, and 8.71-fold enhanced catalytic efficiency for degrading CoA thioesters of cinnamic acid, 4-methoxycinnamic acid, 4-hydroxycinnamic acid, caffeic acid, and ferulic acid, respectively. Molecular dynamics simulation illustrated that novel substrate-binding domains formed strong interaction forces with substrates' methoxy/hydroxyl group and provided hydrophobic/alkaline catalytic surfaces. Five recombinant E. coli with FCS mutants were constructed with the maximum benzaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, protocatechualdehyde, and vanillin productivity of 6.2 ± 0.3, 5.1 ± 0.23, 4.1 ± 0.25, 7.1 ± 0.3, and 8.7 ± 0.2 mM/h, respectively. Hence, our study provided novel and efficient enzymes for the bioconversion of phenylpropanoid acids into aromatic aldehydes.

Functional Characterization of Structural Genomics Proteins in the Crotonase Superfamily.

Members of the Crotonase superfamily, a mechanistically diverse family of proteins that share a conserved quaternary structure, can often catalyze more than one reaction. However, the spectrum of activity for its members has not been well studied. We report on measured crotonase and hydrolase activity for eight structural genomics (SG) proteins from the Crotonase superfamily plus two previously characterized proteins, intended as controls: human enoyl CoA hydratase (ECH) and Anabaena ß-diketone hydrolase. Like most of the 15,000+ SG protein structures deposited in the Protein Data Bank (PDB), the eight SG proteins are of unknown or uncertain biochemical function. The functional characterization of the eight SG proteins is guided by the Structurally Aligned Local Sites of Activity (SALSA), a local-structure-based computational approach to functional annotation. For human ECH, the turnover number for hydrolase activity is threefold higher than that for ECH activity, although the catalytic efficiency is 160-fold higher for ECH. Three SG proteins originally annotated as ECHs were predicted by SALSA to be hydrolases and are observed to have higher catalytic efficiencies for hydrolase activity than for ECH activity, on par with the previously characterized hydrolase. Among the five SG proteins predicted by SALSA to be ECHs, all but one also show some hydrolase activity; all five exhibit lower ECH activity than the human ECH with respect to the crotonyl-CoA substrate. Here, we show examples demonstrating that SALSA can correct functional misannotations even within enzyme families that display promiscuous activity.

Structural insights into bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus.

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3-hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.

A dual cellular-heterogeneous catalyst strategy for the production of olefins from glucose.

Living systems provide a promising approach to chemical synthesis, having been optimized by evolution to convert renewable carbon sources, such as glucose, into an enormous range of small molecules. However, a large number of synthetic structures can still be difficult to obtain solely from cells, such as unsubstituted hydrocarbons. In this work, we demonstrate the use of a dual cellular-heterogeneous catalytic strategy to produce olefins from glucose using a selective hydrolase to generate an activated intermediate that is readily deoxygenated. Using a new family of iterative thiolase enzymes, we genetically engineered a microbial strain that produces 4.3 ± 0.4 g l-1 of fatty acid from glucose with 86% captured as 3-hydroxyoctanoic and 3-hydroxydecanoic acids. This 3-hydroxy substituent serves as a leaving group that enables heterogeneous tandem decarboxylation-dehydration routes to olefinic products on Lewis acidic catalysts without the additional redox input required for enzymatic or chemical deoxygenation of simple fatty acids.

Substrate specificity and conformational flexibility properties of the Mycobacterium tuberculosis ß-oxidation trifunctional enzyme.

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2ß2 tetrameric enzyme. The α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities and the ß-chain provides the 3-ketoacyl-CoA thiolase (KAT) activity. Enzyme kinetic data reported here show that medium and long chain enoyl-CoA molecules are preferred substrates for MtTFE. Modelling studies indicate how the linear medium and long acyl chains of these substrates can bind to each of the active sites. In addition, crystallographic binding studies have identified three new CoA binding sites which are different from the previously known CoA binding sites of the three TFE active sites. Structure comparisons provide new insights into the properties of ECH, HAD and KAT active sites of MtTFE. The interactions of the adenine moiety of CoA with loop-2 of the ECH active site cause a conformational change of this loop by which a competent ECH active site is formed. The NAD+ binding domain (domain C) of the HAD part of MtTFE has only a few interactions with the rest of the complex and adopts a range of open conformations, whereas the A-domain of the ECH part is rigidly fixed with respect to the HAD part. Two loops, the CB1-CA1 region and the catalytic CB4-CB5 loop, near the thiolase active site and the thiolase dimer interface, have high B-factors. Structure comparisons suggest that a competent and stable thiolase dimer is formed only when complexed with the α-chains, highlighting the importance of the assembly for the proper functioning of the complex.

Crystal structure of enoyl-CoA hydratase from Thermus thermophilus HB8.

Fatty-acid degradation is an oxidative process that involves four enzymatic steps and is referred to as the ß-oxidation pathway. During this process, long-chain acyl-CoAs are broken down into acetyl-CoA, which enters the mitochondrial tricarboxylic acid (TCA) cycle, resulting in the production of energy in the form of ATP. Enoyl-CoA hydratase (ECH) catalyzes the second step of the ß-oxidation pathway by the syn addition of water to the double bond between C2 and C3 of a 2-trans-enoyl-CoA, resulting in the formation of a 3-hydroxyacyl CoA. Here, the crystal structure of ECH from Thermus thermophilus HB8 (TtECH) is reported at 2.85 Šresolution. TtECH forms a hexamer as a dimer of trimers, and wide clefts are uniquely formed between the two trimers. Although the overall structure of TtECH is similar to that of a hexameric ECH from Rattus norvegicus (RnECH), there is a significant shift in the positions of the helices and loops around the active-site region, which includes the replacement of a longer α3 helix with a shorter α-helix and 310-helix in RnECH. Additionally, one of the catalytic residues of RnECH, Glu144 (numbering based on the RnECH enzyme), is replaced by a glycine in TtECH, while the other catalytic residue Glu164, as well as Ala98 and Gly141 that stabilize the enolate intermediate, is conserved. Their putative ligand-binding sites and active-site residue compositions are dissimilar.

Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element.

BACKGROUND: Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties. METHODS: We applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site. RESULTS: We solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change. CONCLUSIONS: TSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change. GENERAL SIGNIFICANCE: Those results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

Crystallographic binding studies of rat peroxisomal multifunctional enzyme type 1 with 3-ketodecanoyl-CoA: capturing active and inactive states of its hydratase and dehydrogenase catalytic sites.

The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the ß-oxidation cycle: the 2E-enoyl-CoA hydratase (ECH) and NAD+-dependent 3S-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Šresolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD+ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.

Protein-protein interaction of Rv0148 with Htdy and its predicted role towards drug resistance in Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis resides inside host macrophages during infection and adapts to resilient stresses generated by the host immune system. As a response, M. tuberculosis codes for short-chain dehydrogenases/reductases (SDRs). These SDRs are nicotinamide adenine dinucleotide-reliant oxidoreductases involved in cell homeostasis. The precise function of oxidoreductases in bacteria especially M. tuberculosis were not fully explored. This study aimed to know the detail functional role of one of the oxidoreductase Rv0148 in M. tuberculosis. RESULTS: In silico analysis revealed that Rv0148 interacts with Htdy (Rv3389) and the protein interactions were confirmed using far western blot. Gene knockout mutant of Rv0148 in M. tuberculosis was constructed by specialized transduction. Macrophage cell line infection with this knockout mutant showed increased expression of pro-inflammatory cytokines. This knockout mutant is sensitive to oxidative, nitrogen, redox and electron transport inhibitor stress agents. Drug susceptibility testing of the deletion mutant showed resistance to first-line drugs such as streptomycin and ethambutol and second-line aminoglycosides such as amikacin and kanamycin. Based on interactorme analysis for Rv0148 using STRING database, we identified 220 most probable interacting partners for Htdy protein. In the Rv0148 knockout mutants, high expression of htdy was observed and we hypothesize that this would have perturbed the interactome thus resulting in drug resistance. Finally, we propose that Rv0148 and Htdy are functionally interconnected and involved in drug resistance and cell homeostasis of M. tuberculosis. CONCLUSIONS: Our study suggests that Rv0148 plays a significant role in various functional aspects such as intermediatory metabolism, stress, homeostasis and also in drug resistance.

Insights into the stability and substrate specificity of the E. coli aerobic ß-oxidation trifunctional enzyme complex.

Degradation of fatty acids by the ß-oxidation pathway results in the formation of acetyl-CoA which enters the TCA cycle for the production of ATP. In E. coli, the last three steps of the ß-oxidation are catalyzed by two heterotetrameric α2ß2 enzymes namely the aerobic trifunctional enzyme (EcTFE) and the anaerobic TFE (anEcTFE). The α-subunit of TFE has 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities whereas the ß-subunit is a thiolase with 3-ketoacyl-CoA thiolase (KAT) activity. Recently, it has been shown that the two TFEs have complementary substrate specificities allowing for the complete degradation of long chain fatty acyl-CoAs into acetyl-CoA under aerobic conditions. Also, it has been shown that the tetrameric EcTFE and anEcTFE assemblies are similar to the TFEs of Pseudomans fragi and human, respectively. Here the properties of the EcTFE subunits are further characterized. Strikingly, it is observed that when expressed separately, EcTFE-α is a catalytically active monomer whereas EcTFE-ß is inactive. However, when mixed together active EcTFE tetramer is reconstituted. The crystal structure of the EcTFE-α chain is also reported, complexed with ATP, bound in its HAD active site. Structural comparisons show that the EcTFE hydratase active site has a relatively small fatty acyl tail binding pocket when compared to other TFEs in good agreement with its preferred specificity for short chain 2E-enoyl-CoA substrates. Furthermore, it is observed that millimolar concentrations of ATP destabilize the EcTFE complex, and this may have implications for the ATP-mediated regulation of ß-oxidation in E. coli.

Mycobacterium tuberculosis Exploits a Heterohexameric Enoyl-CoA Hydratase Retro-Aldolase Complex for Cholesterol Catabolism.

Cholesterol catabolism plays an important role in Mycobacterium tuberculosis's (Mtb's) survival and persistence in the host. Mtb exploits three ß-oxidation cycles to fully degrade the side chain of cholesterol. Five cistronic genes in a single operon encode three enzymes, 3-oxo-4-pregnene-20-carboxyl-CoA dehydrogenase (ChsE1-ChsE2), 3-oxo-4,17-pregnadiene-20-carboxyl-CoA hydratase (ChsH1-ChsH2), and 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA retro-aldolase (Ltp2), to perform the last ß-oxidation cycle in this pathway. Among these three enzymes, ChsH1-ChsH2 and Ltp2 form a protein complex that is required for the catalysis of carbon-carbon bond cleavage. In this work, we report the structure of the full length ChsH1-ChsH2-Ltp2 complex based on small-angle X-ray scattering and single-particle electron microscopy data. Mutagenesis experiments confirm the requirement for Ltp2 to catalyze the retro-aldol reaction. The structure illustrates how acyl transfer between enzymes may occur. Each protomer of the ChsH1-ChsH2-Ltp2 complex contains three protein components: a chain of ChsH1, a chain of ChsH2, and a chain of Ltp2. Two protomers dimerize at the interface of Ltp2 to form a heterohexameric structure. This unique heterohexameric structure of the ChsH1-ChsH2-Ltp2 complex provides entry to further understand the mechanism of cholesterol catabolism in Mtb.

Control of ß-Branching in Kalimantacin Biosynthesis: Application of 13 C†NMR to Polyketide Programming.

The presence of ß-branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential ß-branching modifications. We use purified single and multi-domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of ß-branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of 13 C NMR of a single 13 C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl-CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific ß-branch.

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic ß-oxidation trifunctional enzyme complexes.

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid ß-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.

DspI, a putative enoyl-coenzyme A (CoA) hydratase/isomerase, was proposed to be involved in the synthesis of cis-2-decenoic acid (CDA), a quorum sensing (QS) signal molecule in the pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study provided a structural basis for the dehydration reaction mechanism of DspI during CDA synthesis. Structural analysis reveals that Glu126, Glu146, Cys127, Cys131 and Cys154 are important for its enzymatic function. Moreover, we show that the deletion of dspI results in a remarkable decreased in the pyoverdine production, flagella-dependent swarming motility, and biofilm dispersion as well as attenuated virulence in P. aeruginosa PA14. This study thus unravels the mechanism of DspI in diffusible signal factor (DSF) CDA biosynthesis, providing vital information for developing inhibitors that interfere with DSF associated pathogenicity in P. aeruginosa.

Binding of NADP+ triggers an open-to-closed transition in a mycobacterial FabG ß-ketoacyl-ACP reductase.

The ketoacyl-acyl carrier protein (ACP) reductase FabG catalyzes the NADPH/NADH dependent reduction of ß-ketoacyl-ACP substrates to ß-hydroxyacyl-ACP products, the first reductive step in the fatty acid biosynthesis elongation cycle. FabG proteins are ubiquitous in bacteria and are part of the type II fatty acid synthase system. Mining the Mycobacterium smegmatis genome uncovered several putative FabG-like proteins. Among them, we identified M. smegmatis MSMEG_6753 whose gene was found adjacent to MSMEG_6754, encoding a recently characterized enoyl-CoA dehydratase, and to MSMEG_6755, encoding another potential reductase. Recombinantly expressed and purified MSMEG_6753 exhibits ketoacyl reductase activity in the presence of acetoacetyl-CoA and NADPH. This activity was subsequently confirmed by functional complementation studies in a fabG thermosensitive Escherichia coli mutant. Furthermore, comparison of the apo and the NADP+-bound MSMEG_6753 crystal structures showed that cofactor binding induces a closed conformation of the protein. A ΔMSMEG_6753 deletion mutant could be generated in M. smegmatis, indicating that this gene is dispensable for mycobacterial growth. Overall, these results showcase the diversity of FabG-like proteins in mycobacteria and new structural features regarding the catalytic mechanism of this important family of enzymes that may be of importance for the rational design of specific FabG inhibitors.

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation.

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.

Use of Methylmalonyl-CoA Epimerase in Enhancing Crotonase Stereoselectivity.

The use of methylmalonyl-CoA epimerase (MCEE) to improve stereoselectivity in crotonase-mediated biocatalysis is exemplified by the coupling of MCEE, crotonyl-CoA carboxylase reductase and carboxymethylproline synthase in a three-enzyme one-pot sequential synthesis of functionalised C-5 carboxyalkylprolines starting from crotonyl-CoA and carbon dioxide.