Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Zoogloea ramigera I-16 M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP+-linked and D(-)-beta-hydroxybutyryl CoA specific and the other was NAD+-linked and L(+)-isomer specific. The NADP+-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation for beta-hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 micrometer, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 micrometer. The incorporation of [1-14C]acetyl CoA into poly-beta-hydroxybutyrate (PHB) by bacterial crude extract (containing beta-ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of beta-ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium. These findings suggest that, in Z. ramigera I-16M, acetoacetyl CoA is directly reduced to D(-)-beta-hydroxybutyryl CoA by the NADP+-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.
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PMID:An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera. 2 Aug 66

Eight mutants of Alcaligenes eutrophus defective in the intracellular accumulation of poly-beta-hydroxybutyric acid (PHB) were isolated after transposon Tn5 mutagenesis with the suicide vector pSUP5011. EcoRI fragments which harbor Tn5-mob were isolated from pHC79 cosmid gene banks. One of them, PPT1, was used as a probe to detect the intact 12.5-kilobase-pair EcoRI fragment PP1 in a lambda L47 gene bank of A. eutrophus genomic DNA. In six of these mutants (PSI, API, GPI, GPIV, GPV, and GPVI) the insertion of Tn5-mob was physically mapped within a region of approximately 1.2 kilobase pairs in PP1; in mutant API, cointegration of vector DNA has occurred. In two other mutants (GPII and GPIII), most probably only the insertion element had inserted into PP1. All PHB-negative mutants were completely impaired in the formation of active PHB synthase, which was measured by a radiometric assay. In addition, activities of beta-ketothiolase and of NADPH-dependent acetoacetyl coenzyme A (acetoacetyl-CoA) reductase were diminished, whereas the activity of NADPH-dependent acetoacetyl-CoA reductase was unaffected. In all PHB-negative mutants the ability to accumulate PHB was restored upon complementation in trans with PP1. The PHB-synthetic pathway of A. eutrophus was heterologously expressed in Escherichia coli. Recombinant strains of E. coli JM83 and K-12, which harbor pUC9-1::PP1, pSUP202::PP1, or pVK101::PP1, accumulated PHB up to 30% of the cellular dry weight. Crude extracts of these cells had significant activities of the enzymes PHB synthase, beta-ketothiolase, and NADPH-dependent acetoacetyl-CoA reductase. Therefore, PP1 most probably encodes all three genes of the PHB-synthetic pathway in A. eutrophus. In addition to PHB-negative mutants, we isolated mutants which accumulate PHB at a much lower rate than the wild type does. These PHB-leaky mutants exhibited activities of all three PHB-synthetic enzymes; Tn5-mob had not inserted into PP1, and the phenotype of the wild type could not be restored with fragment PP1. The rationale for this mutant type remains unknown.
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PMID:Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. 284 14

1. The enzymes beta-ketothiolase, acetoacetyl-CoA reductase, acetoacetate-succinate CoA-transferase (;thiophorase') and d(-)-3-hydroxybutyrate dehydrogenase have been partially purified from crude extracts of glucose-grown nitrogen-fixing batch cultures of Azotobacter beijerinckii. The condensation of acetyl-CoA to acetoacetyl-CoA catalysed by beta-ketothiolase is inhibited by CoASH, and the reverse reaction is inhibited by acetoacetyl-CoA. Acetoacetyl-CoA reductase has K(m) for acetoacetyl-CoA of 1.8mum and is inhibited by acetoacetyl-CoA above 10mum. The enzyme utilizes either NADH or NADPH as electron donor. The second enzyme of poly-beta-hydroxybutyrate degradation, d(-)-3-hydroxybutyrate dehydrogenase, is NAD(+)-specific and is inhibited by NADH, pyruvate and alpha-oxoglutarate. CoA transferase is inhibited by acetoacetate, the product of hydroxybutyrate oxidation. In continuous cultures poly-beta-hydroxybutyrate biosynthesis ceased on relaxation of oxygen-limitation and the rates in situ of oxygen consumption and carbon dioxide evolution of such cultures increased without a concomitant increase in glucose uptake. 2. On the basis of these and other findings a cyclic mechanism for the biosynthesis and degradation of poly-beta-hydroxybutyrate is proposed, together with a regulatory scheme suggesting that poly-beta-hydroxybutyrate metabolism is controlled by the redox state of the cell and the availability of CoASH, pyruvate and alpha-oxoglutarate. beta-Ketothiolase plays a key role in the regulatory process. Similarities to the pathways of poly-beta-hydroxybutyrate biosynthesis and degradation in Hydrogenomonas are discussed.
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PMID:The regulation of poly-beta-hydroxybutyrate metabolism in Azotobacter beijerinckii. 472 25

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50-80% PHB/cell dry mass when the cells are grown in Luria-Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria-Bertani medium, molecular methods have been applied to provide efficient E. coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E. coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the beta-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter-operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E. coli when grown in Luria-Bertani medium without glucose supplement.
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PMID:High-level poly(beta-hydroxybutyrate) production in recombinant Escherichia coli in sugar-free, complex medium. 760 65

A cosmid gene bank of partially EcoRI-digested genomic DNA from Methylobacterium extorquens IBT no. 6 was screened for DNA fragments restoring polyhydroxyalkanoic-acid (PHA) accumulation in the PHA-negative mutant Alkaligenes eutrophus H16 PHB-4. The M. extorquens PHA-synthase structural gene phaCMex was mapped on a 23-kbp EcoRI fragment by complementation studies, by hybridization experiments with heterologous DNA probes from A. eutrophus H16 encoding for phaA, phaB and phaC and by nucleic acid sequence analysis. Evidence for the presence of genes for a beta-ketothiolase or an acetoacetyl-coenzyme A reductase on this fragment was not obtained. The nucleotide sequence of a 3.7-kbp region was obtained. It contained the entire 1.815-kbp phaCMex plus approximately each 900-bp upstream and downstream of phaCMex.PhaCMex encoded a protein of 605 amino acids with a relative molecular mass (M(r)) of 66742, which exhibited 38.1% amino acid identity with the A. eutrophus PHA synthase. Determination of the N-terminal amino acid sequence of an M(r) 65,000 protein, which was enriched concomitantly with the purification of PHA granules in sucrose gradients, revealed a sequence that was identical with the amino acid sequence deduced from the most probable translation start codon except for a valine, which was obviously removed post-translationally. Enzyme analysis, which was done with the native gene and a phaCMex'-'lacZ fusion gene, gave no evidence for expression of phaCMex in Escherichia coli.
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PMID:Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene. 776 12

Peroxisomal beta-oxidation of linoleic acid and arachidonic acid was depressed when 1-palmitoyl-sn-glycero-3-phosphocholine and microsomes were included in incubations. This reduction was due to the esterification of the substrate into the acceptor by microsomal 1-acyl-sn-glycero-3- phosphocholine acyltransferase. The first cycle of the beta-oxidation of 7,10,13,16-docosatetraenoic acid was independent of 1-acyl-sn-glycero-3-phosphocholine and microsomes. However, when arachidonate was produced it was esterified rather than serving as a substrate for continued beta-oxidation. When arachidonate and linoleate were incubated with peroxisomes alone, 2-trans-4,7,10-hexadecatetraenoic acid and 2-trans-4-decadienoic acid were the respective end products of beta-oxidation. 2-Oxo-8,11-heptadecadienone, a catabolite produced from linoleate, was most likely a nonenzymatic decarboxylation product of 3-oxo-9,12-octadecadienoic acid. In addition to the termination of beta-oxidation by microsomal-peroxisomal communication, our results with linoleate and arachidonate suggest that the reaction catalyzed by 2-trans-4-cis-dienoyl-CoA reductase is the control step in double bond removal. In addition, the beta-ketothiolase step may play a regulatory role in the peroxisomal beta-oxidation of linoleate but not arachidonate or 7,10,13,16-docosatetraenoic acid.
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PMID:Arachidonic acid formed by peroxisomal beta-oxidation of 7,10,13,16-docosatetraenoic acid is esterified into 1-acyl-sn-glycero-3-phosphocholine by microsomes. 803 86

A 3.6-kb EcoRI-SalI fragment of Paracoccus denitrificans DNA hybridized with a DNA probe carrying the poly(3-hydroxyalkanoate) (PHA) synthase gene (phaC) of Alcaligenes eutrophus. Nucleotide sequence analysis of this region showed the presence of a 1,872-bp open reading frame (ORF), which corresponded to a polypeptide with a molecular weight of 69,537. Upstream of the ORF, a promoter-like sequence was found. Escherichia coli carrying the fusion gene between lacZ and the ORF accumulated a level of poly(3-hydroxybutyrate) that was as much as 20 wt% of the cell dry weight in the presence of beta-ketothiolase and acetoacetylcoenzyme A reductase genes of A. eutrophus. The ORF was designated phaCPd. A plasmid vector carrying the phaCPd'-'lacZ fusion gene downstream of the promoter-like sequence expressed beta-galactosidase activity in P. denitrificans. When a multicopy and broad-host-range vector carrying the ORF along with the promoter-like sequence was introduced into P. denitrificans, the PHA content in the cells increased by twofold compared with cells carrying only a vector sequence.
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PMID:Molecular analysis of the poly(3-hydroxyalkanoate) synthase gene from a methylotrophic bacterium, Paracoccus denitrificans. 855 May 12

Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available. The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus. In R. capsulatus, the phaAB genes are not linked to the phaC gene. Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome. Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level. Consistent with this conclusion, it was shown that the R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis. The doubling times of R. capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids. Grown on acetone, caproate, or heptanoate, wild-type R. capsulatus produced high levels of PHAs. Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present. The nutritional versatility and bioenergetic versatility of R. capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production.
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PMID:Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology. 925 Nov 89

Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells. Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks. Biomass doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 days) were significantly higher than those for untransformed cultures (2.01 +/- 0.33 days). Transgenic expression of the bacterial enzymes beta-ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase (0.636 units/mg protein) was detected by enzyme assays and immunoblots. However, over the first 2 years of cultivation, reductase activity decreased to 0.120 units/mg proteins. Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture. These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable. A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation. Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non-polymeric, presumably H-CoA. The synthesis of HB-CoA appeared to be linked to substrate growth limitation, with HB-CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium. This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells.
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PMID:Growth kinetics, nutrient uptake, and expression of the Alcaligenes eutrophus poly(beta-hydroxybutyrate) synthesis pathway in transgenic maize cell suspension cultures. 926 73

The polyhydroxyalkanoate (PHA) synthase gene of Comamonas acidovorans DS-17 (phaCCa) was cloned by using the synthase gene of Alcaligenes eutrophus as a heterologous hybridization probe. Complete sequencing of a 4.0-kbp SmaI-HindIII (SH40) subfragment revealed the presence of a 1,893-bp PHA synthase coding region which was followed by a 1,182-bp beta-ketothiolase gene (phaACa). Both the translated products of these genes showed significant identity, 51.1 and 74.2%, respectively, to the primary structures of the products of the corresponding genes in A. eutrophus. The arrangement of PHA biosynthesis genes in C. acidovorans was also similar to that in A. eutrophus except that the third gene, phaB, coding for acetoacetyl-coenzyme A reductase, was not found in the region downstream of phaACa. The cloned fragment complemented a PHA-negative mutant of A. eutrophus, PHB-4, resulting in poly-3-hydroxybutyrate accumulation of up to 73% of the dry cell weight when fructose was the carbon source. The heterologous expression enabled the incorporation of 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate monomers. The PHA synthase of C. acidovorans does not appear to show any preference for 4-hydroxybutyryl-coenzyme A as a substrate. This leads to the suggestion that in C. acidovorans, it is the metabolic pathway, and not the specificity of the organism's PHA synthase, that drives the incorporation of 4HB monomers, resulting in the efficient accumulation of PHA with a high 4HB content.
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PMID:Genetic analysis of Comamonas acidovorans polyhydroxyalkanoate synthase and factors affecting the incorporation of 4-hydroxybutyrate monomer. 972 94


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