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PubMed2002

A novel plasma membrane quinone reductase and NAD(P)H:quinone oxidoreductase 1 are upregulated by serum withdrawal in human promyelocytic HL-60 cells.

We have studied changes in plasma membrane NAD(P)H:quinone oxidoreductases of HL-60 cells under serum withdrawal conditions, as a model to analyze cell responses to oxidative stress. Highly enriched plasma membrane fractions were obtained from cell homogenates. A major part of NADH-quinone oxidoreductase in the plasma membrane was insensitive to micromolar concentrations of dicumarol, a specific inhibitor of the NAD(P)H:quinone oxidoreductase 1 (NQOI, DT-diaphorase), and only a minor portion was characterized as DT-diaphorase. An enzyme with properties of a cytochrome b5 reductase accounted for most dicumarol-resistant quinone reductase activity in HL-60 plasma membranes. The enzyme used mainly NADH as donor, it reduced coenzyme Q0 through a one-electron mechanism with generation of superoxide, and its inhibition profile by p-hydroxymercuribenzoate was similar to that of authentic cytochrome b5 reductase. Both NQO1 and a novel dicumarol-insensitive quinone reductase that was not accounted by a cytochrome b5 reductase were significantly increased in plasma membranes after serum deprivation, showing a peak at 32 h of treatment. The reductase was specific for NADH, did not generate superoxide during quinone reduction, and was significantly resistant to p-hydroxymercuribenzoate. The function of this novel quinone reductase remains to be elucidated whereas dicumarol inhibition of NQO1 strongly potentiated growth arrest and decreased viability of HL-60 cells in the absence of serum. Our results demonstrate that upregulation of two-electron quinone reductases at the plasma membrane is a mechanism evoked by cells for defense against oxidative stress caused by serum withdrawal.

Animals↗
PubMed1981

Kinetic and physical properties of the L-malate-NAD+ oxidoreductase from Methanospirillum hungatii and comparison with the enzyme from other sources.

The L-malate-NAD+ oxidoreductase of Methanospirillum hungatii was purified to homogeneity by using Blue Sepharose and ADP-Sepharose affinity chromatography. The molecular weight was estimated as 61 700 +/- 1900 by gel filtration and 64 200 +/- 1200 by ultracentrifugation. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein is composed of two polypeptide chains, each corresponding to 31 350 +/- 2150 daltons. Inhibition patterns obtained for malate, alpha-oxoglutarate and ADP established that the sequential reaction mechanism was ordered, with NADH serving as the first substrate. Intracellular concentrations of oxaloacetate approximated the Km value of 27 microM, but NADH was present at less than Km values. Comparison of the amino-acid composition of the L-malate-NAD+ oxidoreductase of M. hungatii and 22 others from prokaryotic and eukaryotic cells revealed a significant direct relationship between average hydrophobicity and the frequency of non-polar side chains, as well as a significant indirect relationship between average hydrophobicity and the polarity ratio. Calculations based on amino-acid-composition data indicated significant composition similarity between pairs of mammalian-cytoplasmic or pairs of mitochondrial L-malate-NAD+ oxidoreductases from various sources, but no significant composition similarity between any of the pairs of bacterial species examined.

Amino Acids↗
PubMed1988

Effects of 5-azacytidine and methyl-group deficiency on NAD(P)H: quinone oxidoreductase and glutathione S-transferase in liver.

Treatment with 5-azacytidine or dietary methyl-group deficiency effected DNA hypomethylation in mouse liver. With these treatments, NAD(P)H: quinone oxidoreductase (EC 1.6.99.2) and some glutathione S-transferase (EC 2.5.1.18) activities were over-expressed, lactate dehydrogenase (EC 1.1.1.27) activity was unaffected and the level of cytochrome P-450 was decreased. The 5-azacytidine induction of NAD(P)H: quinone oxidoreductase was significantly suppressed by puromycin, suggesting that increased enzyme activity results from an elevated level of enzyme-protein synthesis. Regulation at the transcriptional level was revealed by a substantial increase in mRNA of NAD(P)H: quinone oxidoreductase, as shown by Northern-blot analysis. The enzyme pattern observed with 5-azacytidine and with the (carcinogenic) dietary methyl-group deficiency resembles that found in hepatic nodules.

Animals↗
PubMed2000

Structure of the F420H2:quinone oxidoreductase of Archaeoglobus fulgidus identification and overproduction of the F420H2-oxidizing subunit.

The F420H2:quinone oxidoreductase from the sulfate-reducing archaeon Archaeoglobus fulgidus is encoded by the fqo gene cluster which comprises 11 genes (fqo J, K, M, L, N, A, BC, D, H, I, F). The last gene of the cluster, fqoF, was overexpressed in Escherichia coli. The purified subunit was able to oxidize reduced cofactor F420 using the electron-acceptor system methyl viologen plus metronidazole. The specific activity at 78 degrees C was 64 micromol F420H2 oxidized. min-1.(mg protein)-1. The purified polypeptide contained 10.6 mol non-heme iron, 7.2 mol acid-labile sulfur and 0.7 mol FAD per mol protein. With the exception of fqoF, the deduced amino-acid sequences of all other genes show homologies to distinct subunits of NADH-quinone oxidoreductases from prokaryotes and eukaryotes. Thus, it is concluded that the F420H2-dependent and the NADH-dependent enzyme are functional equivalents. Both proteins are the initial enzymes of membrane-bound electron-transport systems and are involved in energy conservation. In parallel with bacterial complex I, the F420H2:quinone oxidoreductase may be composed of three subcomplexes. FqoF functions as the input device adjusted to the oxidation of reduced cofactor F420H2, thereby replacing subunits of the input module of complex I that are not present in A. fulgidus. The subunits FqoB, FqoCD and FqoI may form the membrane-associated module and transfer electrons to the membrane-integral module. It is most likely that the last subcomplex is composed of FqoA, FqoH, FqoJ, FqoK, FqoL, FqoM and FqoN. All subunits are highly hydrophobic and are probably involved in the reduction of a special menaquinone with a fully reduced isoprenoid side chain present in the cytoplasmic membrane of A. fulgidus.

Archaeoglobus fulgidus↗
PubMed1999

Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis.

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.

Alkaline Phosphatase↗
PubMed2000

Sulfite:Cytochrome c oxidoreductase from Thiobacillus novellus. Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family.

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC ). Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alphabeta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa mono-heme cytochrome c(552) subunit (midpoint redox potential, E(m8.0) = +280 mV). The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K(m) values for sulfite and cytochrome c(550) were determined to be 27 and 4 micrometer, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Amino Acid Sequence↗
PubMed2002

Thiol-disulfide oxidoreductases are essential for the production of the lantibiotic sublancin 168.

Thiol-disulfide oxidoreductases are required for disulfide bond formation in proteins that are exported from the cytoplasm. Four enzymes of this type, termed BdbA, BdbB, BdbC, and BdbD, have been identified in the Gram-positive eubacterium Bacillus subtilis. BdbC and BdbD have been shown to be critical for the folding of a protein required for DNA uptake during natural competence. In contrast, no function has been assigned so far to the BdbA and BdbB proteins. The bdbA and bdbB genes are located in one operon that also contains the genes specifying the lantibiotic sublancin 168 and the ATP-binding cassette transporter SunT. Interestingly sublancin 168 contains two disulfide bonds. The present studies demonstrate that SunT and BdbB, but not BdbA, are required for the production of active sublancin 168. In addition, the BdbB paralogue BdbC is at least partly able to replace BdbB in sublancin 168 production. These observations show the unprecedented involvement of thiol-disulfide oxidoreductases in the synthesis of a peptide antibiotic. Notably BdbB cannot complement BdbC in competence development, showing that these two closely related thiol-disulfide oxidoreductases have different, but partly overlapping, substrate specificities.

ATP-Binding Cassette Transporters↗
PubMed2005

The Drosophila ninaG oxidoreductase acts in visual pigment chromophore production.

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaG(P330) mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaG(P330). These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal.

Animals↗
PubMed2004

A male twin infant with skull deformity and elevated neonatal 17-hydroxyprogesterone: a prismatic case of P450 oxidoreductase deficiency.

We report on a male twin infant who presented with brachy-turri-cephaly, frontal bossing, large anterior fontanelle, low set and malformed ears, and mild arachnodactyly. He had normal male genitalia. There was no evidence for maternal virilization during pregnancy. The pattern of malformations resembled Antley-Bixler-Syndrome (ABS). However, sequencing analysis of the fibroblast growth factor receptor 2 gene (FGFR2) did not reveal mutations. The boy's twin sister did not show any somatic or endocrine abnormalities. In the boy, neonatal screening for congenital adrenal hyperplasia was positive with moderately elevated 17-hydroxyprogesterone. Sequence analysis of his CYP21 gene did not reveal any mutations. The short synacthen test revealed an exaggerated 17-hydroxyprogesterone and a blunted cortisol response. Urinary steroid profiling by gas chromatography-mass spectrometry (GC-MS) revealed a unique steroid metabolome suggestive of impaired activity of both 17-hydroxylase and 21-hydroxylase. Clinical and metabolic findings therefore were compatible with the recently described variant of congenital adrenal hyperplasia, P450 oxidoreductase deficiency (ORD). Subsequently, sequencing analysis of CPR, the gene encoding P450 oxidoreductase (OR), revealed a homozygous mutation in the patient, resulting in an amino acid exchange in position 284 of the OR protein (A284P). Both the female twin sister and the parents were heterozygous for the A284P mutation. P450 oxidoreductase deficiency represents a novel autosomal recessively inherited form of congenital adrenal hyperplasia. Its characteristic steroid metabolome can readily be detected by GC-MS analysis of spot urine. Clinical features may include an ABS phenotype, ambiguous genitalia (virilization in girls, feminization in boys), and glucocorticoid deficiency. If required, hydrocortisone replacement should be provided.

17-alpha-Hydroxyprogesterone↗
PubMedDate not supplied

Role of membrane-bound thiol-disulfide oxidoreductases in endospore-forming bacteria.

Thiol-disulfide oxidoreductases catalyze formation, disruption, or isomerization of disulfide bonds between cysteine residues in proteins. Much is known about the functional roles and properties of this class of redox enzymes in vegetative bacterial cells but their involvement in sporulation has remained unknown until recently. Two membrane-embedded thiol-disulfide oxidoreductases, CcdA and StoA/SpoIVH, conditionally required for efficient production of Bacillus subtilis heat-resistant endospores, have now been identified. Properties of mutant cells lacking the two enzymes indicate new aspects in the molecular details of endospore envelope development. This mini-review presents an overview of membrane-bound thiol-disulfide oxidoreductases in the Gram-positive bacterium B. subtilis and endospore synthesis. Accumulated experimental findings on CcdA and StoA/SpoIVH are reviewed. A model for the role of these proteins in endospore cortex biogenesis in presented.

Bacillus subtilis↗
PubMed1994

Reduction of 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233) to a DNA-damaging species: a direct role for NADPH:cytochrome P450 oxidoreductase.

3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, WIN 59075, SR 4233, NSC 130181) has entered phase 1 clinical trials as a bioreductive hypoxic cell cytotoxin because of its novel structure and impressive selective cytotoxicity towards hypoxic cells. Understanding the enzymology and underlying mechanism of oxidative and reductive DNA damage may allow more optimal development and use of this agent and contribute to the rational design of new bioreductive drugs. Here we provide unambiguous evidence that WIN 59075 undergoes one-electron reduction by purified rat liver NADPH:cytochrome P450 oxidoreductase to generate single- and double-strand breaks in plasmid DNA. The DNA damage caused may be important for the therapeutic toxicity of the drug. Enzyme kinetic parameters for this oxidoreductase reaction are in the range 1.01-1.61 mM for Km and 4416-5099 nmol/min/mg for Vmax. The relative levels of expression and cellular localization of target tumour NADPH:cytochrome P450 oxidoreductase may contribute to the therapeutic selectivity of WIN 59075.

Animals↗
PubMed1998

Isolation of a novel NAD(P)H-quinone oxidoreductase from the cyanobacterium Synechocystis PCC6803.

A novel NAD(P)H-quinone oxidoreductase (NQR) was isolated from the cyanobacterium Synechocystis PCC6803 by ion-exchange, affinity and gel-filtration chromatographies. Isolated NQR was found to be a drgA gene product that was a homodimer composed of 23-kDa subunits. It showed NAD(P)H-plastoquinone oxidoreductase activity with Km values for NADPH and NADH of 12 and 48 microM, respectively. The activity was inhibited by thiolmodifying reagents, but not by rotenone, amobarbital, salicylhydroxamic acid, dicumarol, flavone, or diphenyleneiodonium chloride. Therefore, the Cys-147 residue is probably involved in the catalytic reaction. The amino acid sequence of the purified NQR had some homology with those of NADH oxidase, NAD(P)H-flavin oxidoreductase, and nitroreductase but did not contain either an adenine-binding motif or a phosphate-binding motif, thus, it is a new type of NQR.

Amino Acid Sequence↗
PubMed2004

Structure of Escherichia coli YhdH, a putative quinone oxidoreductase.

As part of a structural genomics project on bacterial gene products of unknown function, the crystal structures of YhdH, a putative quinone oxidoreductase, and its complex with NADP have been determined at 2.25 and 2.6 A resolution, respectively. The overall fold of YhdH is very similar to that of alcohol dehydrogenases and quinone reductases despite its low sequence identity. The absence of any Zn ion indicates that YdhH is a putative quinone oxidoreductase. YhdH forms a homodimer, with each subunit composed of two domains: a catalytic domain and a coenzyme-binding domain. NADP is bound in a deep cleft formed between the two domains. Large conformational changes occur upon NADP binding, with the two domains closing up to each other and narrowing the NADP-binding cleft. Comparisons of the YdhH active site with those of the quinone oxidoreductases from Escherichia coli and Thermus thermophilus made it possible to identify essential conserved residues as being Asn41, Asp43, Asp64 and Arg318. The active-site size is very narrow and unless an induced fit occurs is accessible only to reagents the size of benzoquinone.

Amino Acid Sequence↗
PubMed1976

Interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol in rat kidney: heterogeneity of 3alpha-hydroxysteroid oxidoreductases.

3alpha-Hydroxysteroid oxidoreductases catalyzing the interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol (3alpha-androstanediol) have been studied in rat kidney. Three enzymes can be distinguished: a soluble NADPH-dependent oxidoreductase, a microsomal NADPH-dependent enzyme and a microsomal NADH-linked enzyme. Traces of the microsomal enzymes are consistently observed in the 108 000 X g supernatant. Studies on crude preparations reveal that these enzymes differ not only in subcellular localization and co-factor requirement, but also in optimum pH, kinetic characteristics, sensitivity to potential steroidal inhibitors and sensitivity to detergents, ionic strength and temperature. Moreover, salient sex differences exist in the activity of all three kidney enzymes. The soluble NADPH-dependent enzyme is more active in female rats whereas both microsomal enzymes are considerably more active in male animals. The microsomal NADH-dependent oxidoreductase displays favorable characteristics to catalyze the 3alpha-dehydrogenation of 3alpha-androstanediol. Evidence is presented that it is mainly this enzyme that enables the kidney to use 3alpha-androstanediol as an efficient precursor for the local formation of 5alpha-dihydrotestosterone.

Androstane-3,17-diol↗
PubMed1991

The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4.

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.

Animals↗
PubMed1994

F420H2: quinone oxidoreductase from Archaeoglobus fulgidus. Characterization of a membrane-bound multisubunit complex containing FAD and iron-sulfur clusters.

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, was found to contain a membrane-bound F420H2: quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl-beta-D-maltoside the complex was purified 32-fold with a yield of 24%. Using both gel filtration and native PAGE, an apparent molecular mass of approximately 270 kDa was determined. SDS/PAGE revealed the presence of at least seven polypeptides with apparent molecular masses 56, 45, 41, 39, 37, 33, and 32 kDa. The purified complex contained 1.6 mol FAD, 9 mol non-heme iron and 7 mol acid-labile sulfur/mol complex. It did not contain cytochromes, which were, however, present in the membrane fraction of A. fulgidus (3 nmol/mg membrane protein). The purified F420H2: quinone oxidoreductase complex catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone (apparent Km 190 microM) with reduced coenzyme F420 (apparent Km 50 microM) exhibiting a specific activity of 500 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 65 degrees C (temperature optimum). 2-Methyl-1,4-naphthoquinone (menadione), 2-hydroxy-1,4-naphthoquinone, 1,4-naphthoquinone, 2,3-dimethoxy-5-methyl-1,4- benzoquinone, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-ubiquinone) were also reduced with F420H2, albeit with lower rates. The physiological electron acceptor of the F420H2: quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus.

Amino Acid Sequence↗
PubMed1992

Cloning, sequence analysis, and expression of the structural gene encoding glucose-fructose oxidoreductase from Zymomonas mobilis.

The gene encoding glucose-fructose oxidoreductase (gfo) from Zymomonas mobilis was cloned in Escherichia coli and sequenced. An open reading frame of 439 amino acids encoded a protein of 49 kDa. A leader sequence of 52 amino acids preceded the N-terminal sequence of the enzyme, indicating cleavage of the precursor protein at an Ala-Ala site to give rise to an active form of the enzyme of 43 kDa. Processing of the glucose-fructose oxidoreductase leader sequence, although not complete, was demonstrated in an in vitro translation system. The two Z. mobilis promoters of the gfo gene show considerable homology to other highly expressed Z. mobilis genes (pdc, adhB, gap, and pgk) as well as to the E. coli consensus sequence. Although translation of the gfo gene was demonstrated in vitro in an E. coli S30 coupled transcription-translation system, a functional stable protein was not produced in the E. coli clone. However, the gfo gene cloned into a shuttle vector was shown to overexpress glucose-fructose oxidoreductase to levels of up to 6% of the soluble protein in Z. mobilis.

Amino Acid Sequence↗
PubMed2003

2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction.

The conversion of [(14)C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1). 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The alpha-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the gamma-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP(+)-specific KGOR. A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

Acyl Coenzyme A↗