UNIPROT:Q07644 - FACTA Search

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Query: UNIPROT:Q07644 (polypeptide)
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A NADH oxidase has been purified from the extreme thermophile Thermus thermophilus HB8 by several chromatographic steps. The purified enzyme was essentially homogeneous as judged by gel electrophoresis under denaturing conditions and by determination of the N-terminal amino acids sequence. It is a monomeric flavin-adenine-dinucleotide-containing flavoprotein with an apparent molecular mass of 25 kDa and an 1:1 ratio of FAD to the polypeptide chain. The purified enzyme catalyzes the oxidation of reduced NADH or NADPH with the formation of H2O2. The apparent Km values for NADH and NADPH are 4.14 microM and 14.0 microM (pH 7.2 at room temperature), respectively, with a sixfold greater kcat/Km values for NADH compared to NADPH. The enzyme uses O2 as an electron acceptor in the presence of either FAD, riboflavin 5'-phosphate or riboflavin as cofactor. In addition, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (methylene blue, cytochrome c, p-nitroblue tetrazolium, 2,6-dichloroindophenol and potassium ferricyanide), even in the absence of flavin shuttles. No significant inhibition of the NADH oxidoreductase activity by superoxide dismutase was observed with these artificial electron acceptors, indicating that electron transfer occurs mainly from NADH directly to the electron acceptors, not via O2- as an intermediate. The purified NADH oxidase exhibits highest activity at pH 5.0 and is stable at elevated temperatures of up to 80 degrees C.
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PMID:Purification and characterization of a NADH oxidase from the thermophile Thermus thermophilus HB8. 157 5

A highly specific D-hydroxyisovalerate (D-HIV) dehydrogenase, which is a key enzyme in depsipeptide synthesis, was purified to near homogeneity from the enniatin-producing fungus Fusarium sambucinum. The enzyme catalyzes the reversible reaction of 2-ketoisovalerate (2-KIV) to D-HIV. It is strictly dependent on NADPH and exhibits a high substrate specificity with respect to 2-KIV. NADH was not accepted by the enzyme. Km values for 2-KIV and NADPH were found to be 200 and 333 microM, respectively. D-HIV dehydrogenase consists of a single polypeptide chain with a molecular mass of about 53 kDa. Optimum temperature for the reduction of 2-KIV was 35 degrees C and for the oxidation reaction was 45 degrees C. The optimum pH was found to be 7 for the reduction and 8-9 for the oxidation reaction.
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PMID:A highly specific D-hydroxyisovalerate dehydrogenase from the enniatin producer Fusarium sambucinum. 160 49

A full-length cDNA clone encoding barley dihydroflavonol-4-reductase was isolated from a kernel-specific cDNA library by screening with the cDNA of the structural gene (A1) for this enzyme from maize. Subsequently, the gene corresponding to the barley dihydroflavonol-4-reductase cDNA was cloned and sequenced. The gene contains three introns at the same positions as in the Zea mays gene, corresponding to the positions of the first three of the five introns present in the genes of Petunia hybrida and Antirrhinum majus. In vitro transcription and translation of the Hordeum vulgare cDNA clone yielded a protein which converts dihydroquercetin into 2,3-trans-3,4-cis-leucocyanidin with NADPH as cofactor. The protein has a deduced amino acid sequence of 354 residues and a molecular weight of 38,400 daltons. Dihydroflavonol reductases of barley, maize, petunia and snapdragon are highly polymorphic in the NH2- and C-terminal parts of the polypeptide chain while a central region of 324 residues contains 51% identical amino acids. This identity increases to 81% when only the barley and maize enzymes are compared. Recessive mutants in the Ant18 gene tested so far lack dihydroflavonol-4-reductase activity and accumulate small amounts of dihydroquercetin but have retained activity for at least two other enzymes in the flavonoid pathway. In testa-pericarp tissue of mutants ant18-159, ant18-162 and ant18-164, wild-type levels of steady state mRNA for dihydroflavonol reductase have been measured, while mRNA for this enzyme is not transcribed in mutant ant18-161. These data are consistent with the proposal that the Ant18 locus carries the structural gene for dihydroflavonol-4-reductase of barley.
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PMID:Structure of the Hordeum vulgare gene encoding dihydroflavonol-4-reductase and molecular analysis of ant18 mutants blocked in flavonoid synthesis. 172 Aug 64

Two gor genes encoding different mutants of Escherichia coli glutathione reductase have been expressed in the same E. coli cell, leading to the creation of a hybrid form of the enzyme dimer. One of the gor genes carried, in addition to various directed mutations, a 5' extension that encodes a benign penta-arginine "arm" added to the N-terminus of the glutathione reductase polypeptide chain [Deonarain, M.P., Scrutton, N.S., & Perham, R.N. (1992) Biochemistry (preceding paper in this issue)]. This made possible, by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis, the facile separation of the hybrid enzyme from the two parental forms. Moreover, the two subunits in the hybrid enzyme could be made to carry different mutations. In this way, glutathione reductases with only one active site per dimer were generated: the effects of replacing tyrosine-177 with glycine in the NADPH-binding site, which greatly diminishes the Km for glutathione and switches the kinetic mechanism from ping-pong to ordered sequential, and of replacing His-439 with glutamine in the glutathione-binding site, which greatly diminishes the Km for NADPH, were both found to be restricted to the one active site carrying the mutations. This system of generating separable enzyme hybrids is generally applicable and should make it possible now to undertake a more systematic study of catalytic mechanism and assembly for the many enzymes with quaternary structure.
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PMID:Engineering surface charge. 2. A method for purifying heterodimers of Escherichia coli glutathione reductase. 173 9

The XYL1 gene of the yeast Pichia stipitis has been isolated from a genomic library using a specific cDNA probe, and its nucleotide (nt) sequence has been determined. In the 5' noncoding region of the P. stipitis XYL1 gene a TATAAA element (known to be necessary for transcription initiation in most yeast genes) is found at nt -81, and two CCAAT recognition motifs (often referred to as the CCAAT box) are present at nt -146 and -106. The XYL1 encodes a polypeptide of 35,927 Da that constitutes a NADH/NADPH-dependent xylose reductase (XR). The enzyme is part of the xylose-xylulose pathway that is absent or only weakly expressed in Saccharomyces cerevisiae. Extensive homology is found to the N terminus of the XR of Pachysolen tannophilus and Candida shehatae. None of the known cofactor binding domains found in many NAD-dependent dehydrogenases are present in the protein. Transformants of S. cerevisiae containing XYL1 of P. stipitis synthesize an active XR. Fusion of XYL1 with the prokaryotic tac promoter leads to a gene that can be expressed in S. cerevisiae and Escherichia coli.
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PMID:Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis. 175 86

Thioltransferase activity was identified and the enzyme purified to apparent homogeneity from human red blood cells. Activity was measured as glutathione-dependent reduction of the prototype substrate hydroxyethyl disulfide; formation of oxidized glutathione (GSSG) was coupled to NADPH oxidation by GSSG reductase (1 unit of activity = 1 mumol/min of NADPH oxidized). The thioltransferase-GSH-GSSG reductase system was shown also to catalyze the regeneration of hemoglobin from the mixed disulfide hemoglobin-S-S-glutathione (HbSSG) and to reactivate the metabolic control enzyme phosphofructokinase (PFK) after oxidation of its sulfhydryl groups. On a relative concentration basis, thioltransferase was about 1200 times more efficient than dithiothreitol in reactivation of phosphofructokinase; e.g., 500 microM DTT was required to effect the same extent of reactivation as that of 0.4 microM TTase. The GSH plus GSSG reductase system without thioltransferase was ineffective for reduction of HbSSG or reactivation of PFK. The average amount of thioltransferase in intact erythrocytes was calculated to be 4.6 units/g of Hb at 25 degrees C. This level of activity is about the same as those of other enzymes that participate in sulfhydryl maintenance in red blood cells, such as GSSG reductase and glucose-6-phosphate dehydrogenase. These results suggest a physiological role for the thioltransferase in erythrocyte sulfhydryl homeostasis. Certain properties of the human erythrocyte thioltransferase resemble those of other mammalian thioltransferase and glutaredoxin enzymes. Thus, the human erythrocyte enzyme, purified about 28,000-fold to apparent homogeneity, is a single polypeptide with a molecular weight of 11,300. Its N-terminus is blocked, it is heat stable, and it contains four cysteine residues per protein molecule. However, the human erythrocyte thioltransferase is a distinct protein based on its amino acid composition. For example, it contains no methionine residues; whereas the related mammalian enzymes described to date have at least one internal methionine residue in their largely homologous sequences.
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PMID:Thioltransferase in human red blood cells: purification and properties. 182 80

NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate.
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PMID:The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4. 189 80

A novel NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) was purified by sequential fractionation of rat liver cytosol on Q-Sepharose, phenyl-Sepharose, red-Sepharose, and polyacrylamide gel electrophoresis under nondenaturing conditions. The CTBP had a sedimentation coefficient of 5.1S, a Stokes' radius of 35 A, and a calculated mol wt of 76,000. The apparently homogenous protein consisted of a dimer of a polypeptide chain with a mol wt of 38,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADPH increased both the affinity and maximal binding capacity for T3 in the 5.1S CTBP. The maximal activity to bind T3 was obtained by 3.0 x 10(-8) M NADPH. The calculated maximal affinity constant was 2.4 x 10(9) M-1, and the maximal binding capacity was 21,000 pmol T3/mg 5.1S CTBP. The order of affinity of iodothyronine analogs to the 5.1S CTBP was as follows: D-T3 greater than L-T3 greater than L-T4 greater than triiodothyroacetic acid. The optimal pH for T3 binding was 7.2-7.5. Ca2+, Mg2+, and Mn2+ (0.1-10 mM) did not influence T3 binding to CTBP. Zn2+ (1.0 mM), however, inhibited the binding. These results suggested that 5.1S NADPH-dependent CTBP, which is distinct from 4.7S CTBP that had been purified in our laboratory from rat kidney, is present in rat liver.
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PMID:A novel NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine-binding protein (CTBP; 5.1S) in rat liver: a comparison with 4.7S NADPH-dependent CTBP. 191 60

NADPH:isoflavone oxidoreductase (IFR) is the first soluble enzyme of the pterocarpan-specific part of phytoalexin biosynthesis in chickpea (Cicer arietinum L.). The enzyme was purified to apparent homogeneity by a five-step procedure from chickpea cell cultures treated with yeast extract as elicitor. Analysis by gel filtration and SDS/PAGE showed that the enzyme consists of a single polypeptide with a molecular mass of 36 kDa. Km values for the substrates 2'-hydroxyformononetin, 2'-hydroxypseudobaptigenin and NADPH were 6, 6 and 20 microM, respectively. The IFR showed pronounced specificity for the substitution pattern of isoflavones. We found a 2'-hydroxy group and a 4',5'-methylenedioxy or 4'-methoxy function to be essential for acceptance as substrate. The isoelectric point of the protein was determined as 6.3 by IEF and there is no evidence for the existence of isoenzymes. Partial amino acid sequences of IFR were determined from internal peptides obtained by tryptic digestion of the protein and corresponding oligonucleotides were synthesized. A lambda gt10 cDNA library was constructed using poly(A)-rich RNA isolated from chickpea cell cultures treated with Ascochyta rabiei elicitor. 150 positive clones were obtained by screening 2 x 10(5) clones with an IFR-specific oligonucleotide. The identity of sequenced clones was confirmed by comparison of the deduced amino acid sequence with the internal peptide sequences of purified IFR. The sequence of a 1183-bp clone contained a continuous open reading frame of 954 bases encoding a polypeptide of 318 amino acids with a calculated molecular mass of 35.4 kDa, indicating that a full-length cDNA coding for IFR was isolated.
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PMID:Pterocarpan phytoalexin biosynthesis in elicitor-challenged chickpea (Cicer arietinum L.) cell cultures. Purification, characterization and cDNA cloning of NADPH:isoflavone oxidoreductase. 191 47

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction. Extramitochondrial malic enzyme has been isolated from the post-mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, ADP-Sepharose-4B and Sephacryl S-300 to apparent homogeneity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 mumol.min-1.mg protein-1, which corresponds to about 10,000-fold purification. The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is a tetramer composed of identical-molecular-mass subunits. The isoelectric point of the isolated enzyme was 5.65. The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction. The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP-linked decarboxylation reaction varied with malate concentration. The Km values determined at pH 7.2 for malate and NADP were 120 microM and 9.2 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 microM and 27.9 mM, respectively. The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. The Km values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD-dependent decarboxylation reaction was not reversible. The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetate and NADPH-linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP-linked oxidative decarboxylation of malate, respectively. The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.
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PMID:Malic enzyme in human liver. Intracellular distribution, purification and properties of cytosolic isozyme. 193 31

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