Purification and characterization of 2-enoyl-CoA reductase from bovine liver.
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ABSTRACT:
SUBMITTER: Cvetanovic M
PROVIDER: S-EPMC1144807 | biostudies-other | 1985 Apr
REPOSITORIES: biostudies-other
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Project description:The rapid rise in bacterial drug resistance coupled with the low number of novel antimicrobial compounds in the discovery pipeline has led to a critical situation requiring the expedient discovery and characterization of new antimicrobial drug targets. Enzymes in the bacterial fatty acid synthesis pathway, FAS-II, are distinct from their mammalian counterparts, FAS-I, in terms of both structure and mechanism. As such, they represent attractive targets for the design of novel antimicrobial compounds. Enoyl-acyl carrier protein reductase II, FabK, is a key, rate-limiting enzyme in the FAS-II pathway for several bacterial pathogens. The organism, Porphyromonas gingivalis, is a causative agent of chronic periodontitis that affects up to 25% of the US population and incurs a high national burden in terms of cost of treatment. P. gingivalis expresses FabK as the sole enoyl reductase enzyme in its FAS-II cycle, which makes this a particularly appealing target with potential for selective antimicrobial therapy. Herein we report the molecular cloning, expression, purification and characterization of the FabK enzyme from P. gingivalis, only the second organism from which this enzyme has been isolated. Characterization studies have shown that the enzyme is a flavoprotein, the reaction dependent upon FMN and NADPH and proceeding via a Ping-Pong Bi-Bi mechanism to reduce the enoyl substrate. A sensitive assay measuring the fluorescence decrease of NADPH as it is converted to NADP(+) during the reaction has been optimized for high-throughput screening. Finally, protein crystallization conditions have been identified which led to protein crystals that diffract x-rays to high resolution.
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Project description: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.
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Project description:Very long chain fatty acids are important components of plant lipids, suberins, and cuticular waxes. Trans-2-enoyl-CoA reductase (ECR) catalyses the fourth reaction of fatty acid elongation, which is NADPH dependent. In the present study, the expression of two cotton ECR (GhECR) genes revealed by quantitative RT-PCR analysis was up-regulated during cotton fibre elongation. GhECR1 and 2 each contain open reading frames of 933 bp in length, both encoding proteins consisting of 310 amino acid residues. GhECRs show 32% identity to Saccharomyces cerevisiae Tsc13p at the deduced amino acid level, and the GhECR genes were able to restore the viability of the S. cerevisiae haploid tsc13-deletion strain. A putative non-classical NADPH-binding site in GhECR was predicted by an empirical approach. Site-directed mutagenesis in combination with gas chromatography-mass spectrometry analysis suggests that G(5X)IPXG presents a putative novel NADPH-binding motif of the plant ECR family. The data suggest that both GhECR genes encode functional enzymes harbouring non-classical NADPH-binding sites at their C-termini, and are involved in fatty acid elongation during cotton fibre development.
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Project description:Four differently modified forms of acyl-CoA-binding protein (ACBP) were identified in ACBP purified from bovine liver. The majority of the purified ACBP was focused at pH 5.9 in isoelectric focusing and could be shown to be N-acetylated ACBP without any further modifications. Two minor peaks were focused at pH 5.25 and 4.85 respectively. Mass spectrometry and sequence determination showed that the pI 5.25 form was acetylated at Lys18 and that the pI 4.85 form was malonylated in the same position. Furthermore, it could be shown that non-enzymic glycosylation occurred during purification. The acetylated and malonylated variants of ACBP were only found in adult cattle.
| S-EPMC1132611 | biostudies-other
Project description:Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as chronic periodontitis, and exclusively expresses FabK as its enoyl reductase enzyme in the FAS-II pathway. Together, these characteristics distinguish P. gingivalis FabK (PgFabK) as an attractive and novel narrow-spectrum antibacterial target candidate. PgFabK is a flavoenzyme that is dependent on FMN and NADPH as cofactors for the enzymatic reaction, which reduces the enoyl substrate via a ping-pong mechanism. Here, the structure of the PgFabK enzyme as determined using X-ray crystallography is reported to 1.9?Å resolution with endogenous FMN fully resolved and the NADPH cofactor partially resolved. PgFabK possesses a TIM-barrel motif, and all flexible loops are visible. The determined structure has allowed insight into the structural basis for the NADPH dependence observed in PgFabK and the role of a monovalent cation that has been observed in previous studies to be stringently required for FabK activity. The PgFabK structure and the insights gleaned from its analysis will facilitate structure-based drug-discovery efforts towards the prevention and treatment of P. gingivalis infection.
| S-EPMC5947681 | biostudies-literature
Project description:Mitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor ? (PPAR?). However, MECR and PPAR? are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPAR?.To identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPAR?, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPAR? activity.We provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPAR? in the nucleus and increased PPAR?-dependent luciferase activity in HeLa cells.We found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPAR?, and enhances PPAR? activity.
| S-EPMC4091492 | biostudies-literature
Project description:The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.
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Project description:In this study we attempted to determine the number of 2-enoyl-CoA hydratases involved in peroxisomal beta-oxidation. We therefore separated peroxisomal proteins from rat liver on several chromatographic columns and measured hydratase activities on the eluates with different substrates. The results indicate that rat liver peroxisomes contain two hydratase activities: (1) a hydratase activity associated with multifunctional protein 1 (MFP-1) (2-enoyl-CoA hydratase/delta 3, delta 2-enoyl-CoA isomerase/L-3-hydroxyacyl-CoA dehydrogenase) and (2) a hydratase activity associated with MFP-2 (17 beta-hydroxysteroid dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase). MFP-1 forms and dehydrogenates L-3-hydroxyacyl-CoA species, whereas MFP-2 forms and dehydrogenates D-3-hydroxyacyl-CoA species. A portion of MFP-2 is proteolytically cleaved, most probably in the peroxisome, into a 34 kDa 17 beta-hydroxysteroid dehydrogenase/D-3-hydroxyacyl-CoA dehydrogenase and a 45 kDa D-specific 2-enoyl-CoA hydratase. Finally, the results confirm that MFP-1 is involved in the degradation of straight-chain fatty acids, whereas MFP-2 and its cleavage products seem to be involved in the degradation of the side chain of cholesterol (bile acid synthesis).
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Project description:Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van't Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.
| S-EPMC5402281 | biostudies-literature
Project description:The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). How FA metabolic enzymes cooperate with other ER functions, such as calcium (Ca2+) accumulation and its regulated release to the cytosol, is elusive. Trans-2-enoyl-CoA reductase (TER) is a FA reductase present in the ER membrane, and catalyzes the last step of FA elongation cycle and sphingosine degradation pathway. Here we show that TER directly binds to an ER Ca2+ pump, sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b), and regulates its activity. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes. Depletion of TER increases the ER Ca2+ storage and accelerates SERCA2b-dependent Ca2+ uptake to the ER after ligand-induced Ca2+ release. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca2+ accumulation in the ER.
2021-01-13 | PXD020792 | Pride