Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aminoacyl sequences of three regions of pure bovine N1-acetylated polyamine oxidase (PAO) were obtained and used to search GenBankTM. This led to the cloning and sequencing of a complete coding cDNA for murine PAO (mPAO) and the 5'-truncated coding region of the bovine pao (bpao) gene. A search of GenBankTM indicated that mpao maps to murine chromosome 7 as seven exons. The translated amino acid sequences of mpao and bpao have a -Pro-Arg-Leu peroxisomal targeting signal at the extreme C termini. A beta-alpha-beta FAD-binding motif is present in the N-terminal portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to corresponding sections of other flavoprotein amine oxidases, although the overall identity of aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of mpao was used as a probe to establish the relative transcription levels of this gene in various mature murine tissues and murine embryonic and breast tissues at different developmental stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a reasonable level. The mPAO so produced was purified to homogeneity and characterized. It was demonstrated definitively that PAO oxidizes N1-acetylspermine to spermidine and 3-acetamidopropanal and that it also oxidizes N1-acetylspermidine to putrescine and 3-acetamidopropanal. Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that oxidizes these N1-acetylated polyamines on the exo-side of their N4-amino groups. This enzyme is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of the N4-nitrogen. It differs also from mammalian spermine oxidase that oxidizes spermine (but not N1-acetylspermine or N1-acetylspermidine) at the exo-carbon of its N4-amino group. This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state kinetic properties of pure mPAO.
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PMID:Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase. 1266 Feb 32

Enzyme catalyzed biotransformation of the energetic chemical octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is not known. The present study describes a xanthine oxidase (XO) catalyzed biotransformation of HMX to provide insight into the biodegradation pathway of this energetic chemical. The rates of biotransformation under aerobic and anaerobic conditions were 1.6+/-0.2 and 10.5+/-0.9 nmolh(-1)mgprotein(-1), respectively, indicating that anaerobic conditions favored the reaction. The biotransformation rate was about 6-fold higher using NADH as an electron-donor compared to xanthine. During the course of reaction, the products obtained were nitrite (NO(2)(-)), methylenedinitramine (MDNA), 4-nitro-2,4-diazabutanal (NDAB), formaldehyde (HCHO), nitrous oxide (N(2)O), formic acid (HCOOH), and ammonium (NH(4)(+)). The product distribution gave carbon and nitrogen mass-balances of 91% and 88%, respectively. A comparative study with native-, deflavo-, and desulfo-XO and the site-specific inhibition studies showed that HMX biotransformation occurred at the FAD-site of XO. Nitrite stoichiometry revealed that an initial single N-denitration step was sufficient for the spontaneous decomposition of HMX.
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PMID:Mechanism of xanthine oxidase catalyzed biotransformation of HMX under anaerobic conditions. 1280 94

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.
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PMID:2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction. 1452 24

Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with approximately 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H(2) O(2) in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of l-pipecolate to Delta(1)-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [(14)C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite alpha-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.
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PMID:Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis. 1476 47

Nitrate reductase (NR; EC 1.6.6.1-3) catalyzes NAD(P)H reduction of nitrate to nitrite. NR serves plants, algae, and fungi as a central point for integration of metabolism by governing flux of reduced nitrogen by several regulatory mechanisms. The NR monomer is composed of a ~100-kD polypeptide and one each of FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT). NR has eight sequence segments: (a) N-terminal "acidic" region; (b) Mo-MPT domain with nitrate-reducing active site; (c) interface domain; (d) Hinge 1 containing serine phosphorylated in reversible activity regulation with inhibition by 14-3-3 binding protein; (e) cytochrome b domain; (f) Hinge 2; (g) FAD domain; and (h) NAD(P)H domain. The cytochrome b reductase fragment contains the active site where NAD(P)H transfers electrons to FAD. A complete three-dimensional dimeric NR structure model was built from structures of sulfite oxidase and cytochrome b reductase. Key active site residues have been investigated. NR structure, function, and regulation are now becoming understood.
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PMID:NITRATE REDUCTASE STRUCTURE, FUNCTION AND REGULATION: Bridging the Gap between Biochemistry and Physiology. 1501 11

The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN, and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA(3), GA(4), and GA(7) production, but low levels of non-hydroxylated C(20)-GAs (GA(15) and GA(24)) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.
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PMID:The NADPH-cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. 1503 21

Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 A, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the "amine oxidase" class of flavoproteins, such as d-amino acid oxidase and monomeric sarcosine oxidase. With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR(2) subfamily of flavooxidases. On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior. The results do not clarify the metabolic role of this enzyme in B. subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis. However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or d-alanine as carbon or nitrogen sources.
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PMID:Structure-function correlation in glycine oxidase from Bacillus subtilis. 1510 20

The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.
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PMID:Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme. 1511 79

Acyl-CoA dehydrogenases (ACDs) are a family of flavoenzymes that metabolize fatty acids and some amino acids. Of nine known ACDs, glutaryl-CoA dehydrogenase (GCD) is unique: in addition to the alpha,beta-dehydrogenation reaction, common to all ACDs, GCD catalyzes decarboxylation of glutaryl-CoA to produce CO(2) and crotonyl-CoA. Crystal structures of GCD and its complex with 4-nitrobutyryl-CoA have been determined to 2.1 and 2.6 A, respectively. The overall polypeptide folds are the same and similar to the structures of other family members. The active site of the unliganded structure is filled with water molecules that are displaced when enzyme binds the substrate. The structure strongly suggests that the mechanism of dehydrogenation is the same as in other ACDs. The substrate binds at the re side of the FAD ring. Glu370 abstracts the C2 pro-R proton, which is acidified by the polarization of the thiolester carbonyl oxygen through hydrogen bonding to the 2'-OH of FAD and the amide nitrogen of Glu370. The C3 pro-R proton is transferred to the N(5) atom of FAD. The structures indicate a plausible mechanism for the decarboxylation reaction. The carbonyl polarization initiates decarboxylation, and Arg94 stabilizes the transient crotonyl-CoA anion. Protonation of the crotonyl-CoA anion occurs by a 1,3-prototropic shift catalyzed by the conjugated acid of the general base, Glu370. A tight hydrogen-bonding network involving gamma-carboxylate of the enzyme-bound glutaconyl-CoA, with Tyr369, Glu87, Arg94, Ser95, and Thr170, optimizes orientation of the gamma-carboxylate for decarboxylation. Some pathogenic mutations are explained by the structure. The mutations affect protein folding, stability, and/or substrate binding, resulting in inefficient/inactive enzyme.
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PMID:Crystal structures of human glutaryl-CoA dehydrogenase with and without an alternate substrate: structural bases of dehydrogenation and decarboxylation reactions. 1527 22

The NifL regulatory protein controls transcription of nitrogen fixation genes in Azotobacter vinelandii by modulating the activity of the transcriptional activator NifA through direct protein-protein interactions. The ability of NifL to integrate the antagonistic signals of redox and nitrogen status is achieved via the involvement of discrete domains in signalling specific environmental cues. NifL senses the redox status via an FAD co-factor located within the amino-terminal PAS domain and responds to the fixed nitrogen status by interaction with the signal transduction protein GlnK, which binds to the C-terminal GHKL domain of NifL. The GHKL domain binds adenosine nucleotides and is similar to the core catalytic domain of the histidine protein kinases. Binding of ADP to this domain increases the inhibitory activity of NifL and the formation of protein complexes with NifA. This inhibition is antagonised by the binding of 2-oxoglutarate, a key metabolic signal of the carbon status, to the amino-terminal GAF domain of NifA. In this study we have examined the properties of three mutations within conserved residues in the GHKL domain of NifL that impair signal transduction. All three mutations decrease the affinity of NifL for ADP significantly, but the mutant proteins exhibit discrete properties. The N419D mutation prevents inhibition of NifA activity by NifL both in vivo and in vitro. In contrast, the G455A and G480A mutations eliminate the redox response, but the mutant proteins retain some sensitivity to the fixed nitrogen status and the ability to interact with the GlnK signal transduction protein. Our data suggest that the absence of the redox switch in the G455A and G480A mutants is a consequence of their inability to override the allosteric effect of 2-oxoglutarate on NifA activity. Overall, these results demonstrate that the binding of adenosine nucleotides to the GHKL domain of NifL plays an important role in counteracting the response of NifA to 2-oxoglutarate, under conditions that are inappropriate for nitrogen fixation.
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PMID:Mutational analysis of the nucleotide-binding domain of the anti-activator NifL. 1570 8


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