Purification and Properties of 5-Ketogluconate Reductase fromGluconobacter liquefaciens
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5-Ketogluconate reductase (5KGR) from the cell free extract of Gluconobacter liquefaciens (IFO 12388) was partially purified about 120-fold by a procedure employing ammonium sulfate fractionation, and DEAE-cellulose-, hydroxylapatite- and DEAE-Sephadex A-50-column chromatographies. NADP was specifically required for the oxidative reaction of gluconic acid. The optimum pH for the oxidation of gluconic acid (GA) to 5-ketogluconic acid (5KGA) by the enzyme was 10.0 and for the reduction of 5KGA was 7.5. The optimum temperature of the enzyme was 50°C for both reactions of oxidation and reduction. The enzyme was considerably unstable and lost all of its activity within 3 days. The enzyme activity was strongly inhibited with p-chloromercuribenzoate and mercury ion, but remarkably stimulated by EDTA (1 × 10−3m). Apparent Km values were 1.8 × 10−2m for GA, 0.9 × 10−3m for 5KGA, 1.6 × 10−5 m for NADP, and 1.1 × 10−5 m for NADPH2.Coenzyme A
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2-Enoyl-CoA reductase was purified 150-fold from the crude extract of Mycobacterium smegmatis. The purified reductase required NADH, but not NADPH, as a reductant and catalyzed the reduction of C4 to C16 enoyl-CoAs, though the activities toward shorter chain substrates (c4 and C6) were very low. Thiolase was also partially purified from the same source. These two enzymes were used, together with 3-hydroxyacyl-CoA dehydrogenase and the two forms of enoyl-CoA hydratase (hydratases I and II) previously purified from the same source, to reconstitute fatty acid elongation activity. The products formed from [1-14C]acetyl-CoA and decanoyl-CoA in this reconstituted system were analyzed by thin-layer chromatography and radio-gas-liquid chromatography. The system containing hydratase II produced laurate and 3-hydroxylaurate (in the form of their CoA esters) and the ratio of laurate to 3-hydroxylaurate increased as the incubation time was increased. The system containing hydratase I produced only 3-hydroxylaurate. 3-Hydroxylaurate was also the only product when enoyl-CoA reductase was omitted from the system containing hydratase II. It is concluded that hydratase II, but not hydratase I, is functional in fatty acid elongation by M. smegmatis and that enoyl-CoA reductase is also essential for the reaction. CoA and NAD+ inhibited the reconstituted elongation activity in competition with acetyl-CoA and NADH, respectively.
Mycobacterium smegmatis
Thiolase
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The microsomal enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is subject to rapid degradation when cells are incubated with sterols or mevalonic acid (MVA). It has been shown that this rapid degradation is dependent upon both a sterol and another MVA-derived metabolite (Nakanishi, M., Goldstein, J. L., and Brown, M. S. (1988) J. Biol. Chem. 258, 8929-8937). In the current study, inhibitors of the isoprene biosynthetic pathway were used to define further this mevalonic acid derivative involved in the accelerated degradation of HMG-CoA reductase. The accelerated degradation of HMG-CoA reductase in met-18b-2 cells, which is induced by the addition of MVA, was inhibited by the presence of the squalene synthase inhibitor, zaragozic acid/squalestatin, or the squalene epoxidase inhibitor, NB-598. Accelerated degradation of HMG-CoA reductase was observed when NB-598-treated cells were incubated with both MVA and sterols. In contrast, the addition of MVA and sterols to zaragozic acid/squalestatin-treated cells did not result in rapid enzyme degradation. This MVA- and sterol-dependent degradation of HMG-CoA reductase persisted in cells permeabilized with reduced streptolysin O. Finally, the selective degradation of HMG-CoA reductase was also observed in rat hepatic microsomes incubated in vitro in the absence of ATP and cytosol. We conclude that the MVA-derived component that is required for the accelerated degradation of HMG-CoA reductase is derived from farnesyl disphosphate and/or squalene in the isoprenoid biosynthetic pathway. We propose that this component has a permissive effect and does not, by itself, induce the degradation of HMG-CoA reductase. We also conclude that the degradation of HMG-CoA occurs in the endoplasmic reticulum, and, once the degradation of HMG-CoA reductase has been initiated by MVA and sterols, all necessary components for the continued degradation of HMG-CoA reductase reside in the endoplasmic reticulum.
Mevalonic acid
Coenzyme A
Degradation
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Determinations of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity in haploid strains and diploid hybrids of wild-type Saccharomyces cerevisiae revealed that a genetic basis exists for control of this key regulatory enzyme in which low enzyme activity is phenotypically dominant to high enzyme activity. These observations suggested the existence of an inhibitor of reductase activity or a suppressor of enzyme synthesis. Feeding studies using an early sterol intermediate (mevalonolactone) and end-product sterol (ergosterol) indicated that a secondary regulatory site in this pathway operates to decrease the activity of HMG-CoA reductase. This diminution of activity was paralleled by increases in the accumulation of squalene, suggesting that this intermediate (or another isoprenoid derivative) may also play a significant role in the in vivo regulation of sterol biosynthesis. Lastly, feedback inhibition of HMG-CoA reductase by ergosterol was demonstrated in a yeast mutant which is permeable to this sterol. These studies showed that yeast can serve as a eukaryotic model system for a combined biochemical and genetic investigation into the factors which control the activity of HMG-CoA reductase.
Ergosterol
Coenzyme A
Mevalonate pathway
Hydroxymethylglutaryl-CoA reductase
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D-galacturonate reductases are catalysing the reversible reduction of D-galacturonate to L-galactonate using NAD(P)H as a cofactor. The enzymes are part of two different pathways. One pathway is the fungal pathway for the catabolism of the main compound of pectin, D-galacturonate. The other pathway is a a pathway in plants for L-ascorbic acid synthesis. The previously described naturally occurring enzymes preferably use NADPH as a cofactor. Although certain D-galacturonate reductases, such as the reductases from Aspergillus niger or Euglena gracilis also accept NADH, their activity is significantly higher with NADPH. We identified in E. gracilis a gene, called gaa1, coding for a D-galacturonate reductase with similar activities with NADH and NADPH. It is potentially useful for the metabolic engineering of microbes to make use of pectin rich biomass.
Catabolism
Metabolic pathway
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Reduction of enoyl–acyl‐carrier‐protein (ACP) substrates by enoyl‐ACP reductase is a key regulatory step in fatty acid elongation of Escherichia coli. Two enoyl‐ACP reductase activities have been described in E. coli , one specific for NADH, the other for NADPH as cofactor. Because of their distinct enzymatic properties, these activities were ascribed to two different proteins. The NADH‐dependent enoyl‐ACP reductase of E. coli has previously been identified as the FabI protein, which is the target of a group of antibacterial compounds, the diazaborines. We now demonstrate that both enoyl‐ACP reductase activities reside in FabI. In crude cell extracts of FabI‐overproducing strains, both NADH‐dependent and NADPH‐dependent enoyl‐ACP reductase activities are increased. Mutations in the fabI gene that lead either to temperature‐sensitive growth or diazaborine resistance result in the reduction of both activities. When FabI is purified in pH 6.5 buffers, the protein exhibits NADH‐dependent and NADPH‐dependent reductase activities. Both enzymatic activities are inhibited by diazaborine. The NADPH‐dependent enoyl‐ACP reductase activity, however, turned out to be approximately eight times more resistant to diazaborine. The difference in sensitivity indicates that binding of either NADPH or NADH to FabI results in distinct changes in the configuration of the protein or, alternatively, it is different due to the different charge of the cofactors. These effects might be responsible for the differences in the enzymatic properties. Both reductase activities of the FabI protein are inhibited by physiologically relevant concentrations of palmitoyl‐CoA, which might be important in regulating endogenous fatty acid biosynthesis in E. coli in the presence of exogenous fatty acids.
Acyl carrier protein
Coenzyme A
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The occurrence of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase in adult Hymenolepis diminuta was demonstrated. This activity was negligible in the cestode's cytosolic fraction but was noted when the mitochondrial or microsomal fraction served as the enzyme source. The predominant localization of HMG-CoA reductase activity was with the microsomal fraction. This fraction did not contain appreciable mitochondrial contamination based on the distribution of marker enzymes. The enzymatic nature of HMG-CoA conversion to mevalonic acid by either fraction was apparent because the reaction was heat labile and responded linearly to time of assay and protein content. The enzymatic reduction of HMG-CoA absolutely required NADPH when either fraction was assayed. The lesser activity of the mitochondrial fraction was membrane-associated. The predominant localization of HMG-CoA reductase activity with microsomal membranes and its separation with the membranous component of the mitochondrial fraction suggest that mitochondrial activity reflects the presence of microsomal membranes. In its predominant localization and pyridine nucleotide requirement, the cestode's HMG-CoA reductase activity resembles that of mammalian systems. The finding of HMG-CoA reductase provides an enzymatic mechanism for the intermediate conversion of HMG-CoA to mevalonic acid that would be needed for acetate-dependent isoprenoid lipid synthesis by adult H. diminuta.
Hymenolepis diminuta
Mevalonic acid
Coenzyme A
Hydroxymethylglutaryl-CoA reductase
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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.
Flavoprotein
Acyl carrier protein
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Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.
Jerusalem artichoke
Polyclonal antibodies
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