Substrate specificities of the enzymes of the oleate desaturase system from photosynthetic tissue.
Ontology highlight
ABSTRACT:
SUBMITTER: Murphy DJ
PROVIDER: S-EPMC1144580 | biostudies-other | 1985 Jan
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.
| S-EPMC7170512 | biostudies-literature
Project description:Polyunsaturated fatty acids (PUFAs) have important industrial, physiological, and nutritional properties. Plants use the sequential activities of FAD2 and FAD3 desaturases to convert 18:1?9 to the important PUFA 18:3?9,12,15, whereas the fungus Fusarium verticillioides 7600 uses the bifunctional desaturase Fm1 for both reactions. Here, we used a combination of sequence comparisons, structural modeling, and mutagenesis experiments to investigate Fm1's regioselectivity and identified two functionally relevant clusters of residues that contribute to Fm1 activity. We found that cluster I (Leu153, Phe157, and His194), located near the catalytic iron ions, predominantly affects activity, whereas cluster II (Tyr280, His284, and Leu287), located in a helix forming the entrance to the substrate-binding pocket, mainly specifies 15-desaturation. Individual or combined substitutions of cluster II residues substantially reduced 15-desaturation. The combination of F157W from cluster I with Y280L, H284V, and L287T from cluster II created an increased-activity variant that almost completely lost the ability to desaturate at C15 and acted almost exclusively as a 12-desaturase. No variants were identified in which 15-desaturation occurred in the absence of 12-desaturation. Fm1 displayed only traces of activity with C16 substrate, but several cluster I variants exhibited increased activity with both 18:1 and 16:1 substrates, converting 16:1?9 to 16:3?9,12,15, consistent with Fm1 performing sequential v + 3 desaturation reactions at C12 and then C15. We propose that cluster II residues interact with the substrate headgroup when the acyl chain contains both ?9 and ?12 double bonds, in which case C15 becomes positioned adjacent to the di-iron site enabling a second v + 3 desaturation.
| S-EPMC6314118 | biostudies-literature
Project description:Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.
| S-EPMC7306040 | biostudies-literature
Project description:Threonine aldolases have emerged as a powerful tool for asymmetric carbon-carbon bond formation. These enzymes catalyse the unnatural aldol condensation of different aldehydes and glycine to produce highly valuable ?-hydroxy-?-amino acids with complete stereocontrol at the ?-carbon and moderate specificity at the ?-carbon. A range of microbial threonine aldolases has been recently recombinantly produced by several groups and their biochemical properties were characterized. Numerous studies have been conducted to improve the reaction protocols to enable higher conversions and investigate the substrate scope of enzymes. However, the application of threonine aldolases in organic synthesis is still limited due to often moderate yields and low diastereoselectivities obtained in the aldol reaction. This review briefly summarizes the screening techniques recently applied to discover novel threonine aldolases as well as enzyme engineering and mutagenesis studies which were accomplished to improve the catalytic activity and substrate specificity. Additionally, the results from new investigations on threonine aldolases including crystal structure determinations and structural-functional characterization are reviewed.
| S-EPMC4761611 | biostudies-literature
Project description:The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the ?-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities.
| S-EPMC4692089 | biostudies-literature
Project description:1. When [1-14C]oleoyl-CoA was incubated with a pea-leaf homogenate oleate was both incorporated into microsomal 3-sn-phosphatidylcholine and released as the unesterified fatty acid. The proportion of oleate incorporated into this phospholipid was dependent on the relative amounts of thiol ester and microsomal preparation present in reactions. 2. At the concentrations of microsomal preparation and [14C]oleoyl-CoA used to study oleate desaturation the metabolism of the thiol ester was essentially complete after 5 min incubation, but the loss of label from 3-sn-phosphatidylcholine oleate and the concomitant increase in radioactivity in the linoleate of this phospholipid proceeded at approximately linear rates over a 60 min period. The kinetics of labelling of unesterified linoleate was consistent with the view that this labelled fatty acid was derived from 3-sn-phosphatidylcholine. 3. Oleate desaturation required oxygen and with unwashed microsomal fractions was stimulated either by NADPH or by the 105 000g supernatant. Washed microsomal preparations did not catalyse desaturation, but actively was restored by the addition of NADPH, 105 000G supernatant or Sephadex-treated supernatant. NADPH could be replaced by NADH or NADP+, but not by NAD+. 4. Microsomal fractions from mature and immature maize lamina and expanding spinach leaves also rapidly incorporated oleate from ([14C]oleoyl-CoA into 3-sn-phosphatidylcholine, but desaturation of 3-sn-phosphatidylcholine oleate was detected only with microsomal preparations from immature maize lamina. 5. It is proposed that leaf microsomal preparations posses an oleate desaturase for which 3-sn-phosphatidylcholine oleate is either the substrate or an immediate precursor of the substrate.
| S-EPMC1172803 | biostudies-other
Project description:Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
| S-EPMC5784979 | biostudies-literature
Project description:Adipose inflammation is crucial to the pathogenesis of metabolic disorders. This study aimed at identify the effects of stearoyl-CoA desaturase-1 (SCD1) on the inflammatory response of a paracrine network involving adipocytes, macrophages, and endothelial cells.Loss of SCD1 in both genetic (Agouti) and diet-induced obesity (high-fat diet) mouse models prevented inflammation in white adipose tissue and improved its basal insulin signaling. In SCD1-deficient mice, white adipose tissue exhibited lower inflammation, with a reduced response to lipopolysaccharide in isolated adipocytes, but not in peritoneal macrophages. Mimicking the in vivo paracrine regulation of white adipose tissue inflammation, SCD1-deficient adipocyte-conditioned medium attenuated the induction of tumor necrosis factor (TNF) alpha/interleukin 1beta gene expression in RAW264.7 macrophages and reduced the adhesion response in endothelial cells. We further demonstrated that the adipocyte-derived oleate (18:1n9), but not palmitoleate (16:1n7), mediated the inflammation in macrophages and adhesion responses in endothelial cells.Loss of SCD1 attenuates adipocyte inflammation and its paracrine regulation of inflammation in macrophages and endothelial cells. The reduced oleate level is linked to the inflammation-modulating effects of SCD1 deficiency.
| S-EPMC2837593 | biostudies-literature
Project description:BACKGROUND:Trypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites. METHODOLOGY:Cultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied. PRINCIPAL FINDINGS:Growth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC(50) values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC(50) values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential. CONCLUSIONS/SIGNIFICANCE:As OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.
| S-EPMC2997783 | biostudies-literature
Project description:On incubation of microsomal fraction from pea (Pisum sativum L.) leaves with ammonium [1-14C]oleate or [1-14C]oleoyl-CoA in the presence of ATP, CoA, Mg2+ and NADH, the major reactions observed were those catalysed by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. The reaction catalysed by oleoyl phosphatidylcholine desaturase was specifically inhibited by H2O2, and this inhibitory effect was overcome by catalase (EC 1.11.1.6).
| S-EPMC1152115 | biostudies-other