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 nitrate reductase (NR) gene niaA of the oomycete Phytophthora infestans was selected from a gene library by heterologous hybridization. NiaA occurs as a single-copy gene ant its expression is regulated by the nitrogen source. The nucleotide sequence of niaA was determined and comparison of the deduced amino-acid sequence of 902 residues with NRs of higher fungi and plants revealed a significant homology, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The P. infestans niaA gene was used as a model gene to test whether oomycete genes are functional in the ascomycete Aspergillus nidulans, a fungus which is highly accessible for molecular genetic studies. The complete niaA gene was stably integrated into the genome of a nia- deletion mutant of A. nidulans. However, transformants containing one or more copies of the niaA gene were not able to complement the nia- mutant. This suggests that there is no functional expression of the introduced niaA gene in A. nidulans. In addition, the activity of two other oomycete gene promoters was analyzed in a transient expression assay. Plasmids containing chimaeric genes with the promoter of the P. infestans ubiquitin gene ubi3R, or the Bremia lactucae ham34 gene, fused to the coding sequence of the Escherichia coli beta-glucuronidase (GUS) reporter gene, were transferred to A. nidulans protoplasts. No significant GUS activity was detectable indicating that the ubi3R and ham34 promoters are not active in A. nidulans. Apparently, the regulatory sequences which are sufficient for gene activation in oomycetes are not functional in the ascomycete A. nidulans.
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PMID:NiaA, the structural nitrate reductase gene of Phytophthora infestans: isolation, characterization and expression analysis in Aspergillus nidulans. 761 59

Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.
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PMID:The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch. 778 4

Pyrrole-2-carboxylate oxygenase was purified 8.2-fold to homogeneity from Arthrobacter strain Py1 grown on pyrrole-2-carboxylate as sole carbon, nitrogen, and energy source. FAD and dithioerythritol had to be present during the purification procedure to stabilize the enzyme activity. The molecular mass of the pyrrole-2-carboxylate oxygenase was about 160 kDa by gel filtration chromatography and native gradient PAGE, only one polypeptide of about 60 kDa was present after SDS-PAGE. The FAD content was 2.7 to 3.6 mol FAD per enzyme (160 kDa). The non-covalently bound FAD of the pyrrole-2-carboxylate oxygenase was reduced by NADH and reoxidized by oxygen and pyrrole-2-carboxylate. The enzyme exhibited a narrow substrate specificity. Besides pyrrole-2-carboxylate, only pyrrole, pyrrole-2-aldehyde, and indole-2-carboxylate stimulated the oxygen consumption at a very low rate. The enzyme activity was strongly reduced by different sulfhydryl group inhibitors, but it could be restored by 2-mercaptoethanol or dithiothreitol. The content of pyrrole-2-carboxylate oxygenase was about 6% of the soluble protein as determined by antibodies raised against the enzyme. No cross reacting material was present in other bacteria also able to degrade pyrrole-2-carboxylate. A low amount of the enzyme was present in uninduced cells of Arthrobacter strain Py1, although the enzymatic activity was below the detection limit. The N-terminal amino acid sequence of the enzyme did not contain the consensus sequence GXGXXG found to be present close to the N-terminus of many flavin-dependent monoxygenases sequenced so far.
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PMID:Purification and characterization of a pyrrole-2-carboxylate oxygenase from Arthrobacter strain Py1. 801 Nov 78

Three isozymes of nitric oxide synthase (NOS) have been identified. Their cDNA- and protein structures as well as their genomic DNA structures have been described. NOS I (ncNOS, originally discovered in neurons) and NOS III (ecNOS, originally discovered in endothelial cells) are low output, Ca(2+)-activated enzymes whose physiological function is signal transduction. NOS II (iNOS, originally discovered in cytokine-induced macrophages) is a high output enzyme which produces toxic amounts of NO that represent an important component of the antimicrobial, antiparasitic and antineoplastic activity of these cells. Depending on the species, NOS II activity is largely (human) or completely (mouse and rat) Ca(2+)-independent. In the human species, the NOS isoforms I, II and III are encoded by three different genes located on chromosomes 12, 17 and 7, respectively. The amino acid sequences of the three human isozymes (deduced from the cloned cDNAs) show less than 59% identity. Across species, amino acid sequences are more than 90% conserved for NOS I and III, and greater 80% identical for NOS II. All NOS produce NO by oxidizing a guanidino nitrogen of L-arginine utilizing molecular oxygen and NADPH as co-substrates. All isoforms contain FAD, FMN and heme iron as prosthetic groups and require the cofactor BH4. NOS I and III are constitutively expressed in various cells. Nevertheless, expression of these isoforms is subject to regulation. Expression is enhanced by e.g. estrogens (for NOS I and III), shear stress, TGF-beta 1, and (in certain endothelial cells) high glucose (for NOS III). TNF-alpha reduces the expression of NOS III by a post-transcriptional mechanism destabilizing the mRNA. The regulation of the NOS I expression seems to be very complex as reflected by at least 8 different promoters transcribing 8 different exon 1 sequences which are expressed differently in different cell types. Expression of NOS II is mainly regulated at the transcriptional level and can be induced in many cell types with suitable agents such as LPS, cytokines, and other compounds. Whether some cells can express NOS II constitutively is still under debate. Pathways resulting in the induction of the NOS II promoter may vary in different cells. Activation of transcription factor NF-kappa B seems to be an essential step for NOS II induction in most cells. The induction of NOS II can be inhibited by a wide variety of immunomodulatory compounds acting at the transcriptional levels and/or post-transcriptionally.
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PMID:Nitric oxide synthase: expression and expressional control of the three isoforms. 853 63

Polyamine oxidase (PAO) is a FAD-dependent enzyme with a molecular mass of about 62 kDa, present with high activity in most tissues of vertebrates. Structural requirements of a substrate for PAO are two positively charged amino groups, separated by a short carbon chain and an alkyl substituent on one or both nitrogen atoms. Spermine and the monoacetyl derivatives N1-acetylspermine and N1-acetylspermidine appear to be the natural substrates. Spermidine is only poorly oxidized by PAO. Using O2, the substrates are oxidatively cleaved by PAO to form equimolar amounts of an amine, an aldehyde and hydrogen peroxide. PAO is an integral part of the polyamine interconversion cycle, a major intracellular regulatory system, which contributes to the maintenance of polyamine homeostasis in non-proliferating cells, including brain cells. Selective inactivators were used as tools in the elucidation of the functions of PAO. Interestingly, even long-term inactivation of PAO did not provoke behavioral changes in experimental animals, despite considerable changes in polyamine metabolism. PAO inactivation, however, improves the growth-inhibitory effects of inhibitors of polyamine biosynthetic enzymes and the antitumoral effects of some structural analogs of the polyamines.
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PMID:Polyamine oxidase, properties and functions. 858 70

Xanthine dehydrogenase (XDH) is induced in Comamonas acidovorans cells incubated in a limited medium with hypoxanthine as the only carbon and nitrogen source. The enzyme has been purified to homogeneity using standard techniques and characterized. It contains two subunits with M(r) values of 90 and 60 kDa. Gel filtration studies show the enzyme to have an alpha 2 beta 2 native structure. No precursor form of the enzyme is observed on Western blot analysis of cell extracts obtained at various stages of enzyme induction. Metal analysis of the purified enzyme shows 1.1 Mo, 4.0 Fe, and 3.6 phosphorus atoms per alpha beta protomer. Cofactor analysis shows the enzyme to contain a single molybdopterin mononucleotide and one FAD per alpha beta protomer. Electron spin resonance and circular dichroism spectral studies of the oxidized and reduced forms of the enzyme suggest the Fe centers to be two nonidentical [2Fe-2S] clusters. Electron spin resonance signals due to Mo(V) and neutral FAD radical are also observed in the reduced form of the enzyme. Purified enzyme preparations ranged from 70% to 100% functionality. The enzyme is irreversibly inactivated by CN- and is inhibited on incubation with allopurinol. With xanthine and NAD+ as substrates the enzyme has a specific activity of 50 units/mg, a kcat value of 120 s-1, an activity/flavin ratio of 1930, and respective Km values of 66 and 160 mM. Using 8-D-xanthine as substrate, a DV value of 1.8 is found with no change in Km. Thus, the Km and KD values of the enzyme for xanthine are equal. These data show Comamonas XDH to exhibit structural properties similar to bovine milk xanthine oxidase/dehydrogenase and to chicken liver xanthine dehydrogenase. Although the bacterial enzyme exhibits a 6-7-fold greater turnover rate than bovine or avian enzymes, the catalytic efficiencies (as measured by V/K) are similar for all three enzymes.
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PMID:Purification and characterization of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans. 861 34

The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.
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PMID:Characterization of the Aspergillus parasiticus niaD and niiA gene cluster. 866 12

The NIFL regulatory protein controls transcriptional activation of nitrogen fixation (nif) genes in Azotobacter vinelandii by direct interaction with the enhancer binding protein NIFA. Modulation of NIFA activity by NIFL, in vivo occurs in response to external oxygen concentration or the level of fixed nitrogen. Spectral features of purified NIFL and chromatographic analysis indicate that it is a flavoprotein with FAD as the prosthetic group, which undergoes reduction in the presence of sodium dithionite. Under anaerobic conditions, the oxidized form of NIFL inhibits transcriptional activation by NIFA in vitro, and this inhibition is reversed when NIFL is in the reduced form. Hence NIFL is a redox-sensitive regulatory protein and may represent a type of flavoprotein in which electron transfer is not coupled to an obvious catalytic activity. In addition to its ability to act as a redox sensor, the activity of NIFL is also responsive to adenosine nucleotides, particularly ADP. This response overrides the influence of redox status on NIFL and is also observed with refolded NIFL apoprotein, which lacks the flavin moiety. These observations suggest that both energy and redox status are important determinants of nif gene regulation in vivo.
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PMID:Azotobacter vinelandii NIFL is a flavoprotein that modulates transcriptional activation of nitrogen-fixation genes via a redox-sensitive switch. 870 Aug 99

Nitrate is a significant nitrogen source for plants and microorganisms. Recent molecular genetic analyses of representative bacterial species have revealed structural and regulatory genes responsible for the nitrate-assimilation phenotype. Together with results from physiological and biochemical studies, this information has unveiled fundamental aspects of bacterial nitrate assimilation and provides the foundation for further investigations. Well-studied genera are: the cyanobacteria, including the unicellular Synechococcus and the filamentous Anabaena; the gamma-proteobacteria Klebsiella and Azotobacter; and a Gram-positive bacterium, Bacillus. Nitrate uptake in most of these groups seems to involve a periplasmic binding protein-dependent system that presumably is energized by ATP hydrolysis (ATP-binding cassette transporters). However, Bacillus may, like fungi and plants, utilize electrogenic uptake through a representative of the major facilitator superfamily of transport proteins. Nitrate reductase contains both molybdenum cofactor and an iron-sulfur cluster. Electron donors for the enzymes from cyanobacteria and Azotobacter are ferredoxin and flavodoxin, respectively, whereas the Klebsiella and Bacillus enzymes apparently accept electrons from a specific NAD(P)H-reducing subunit. These subunits share sequence similarity with the reductase components of bacterial aromatic ring-hydroxylating dehydrogenases such as toluene dioxygenase. Nitrite reductase contains sirohaem and an iron-sulfur cluster. The enzymes from cyanobacteria and plants use ferredoxin as the electron donor, whereas the larger enzymes from other bacteria and fungi contain FAD and NAD(P)H binding sites. Nevertheless, the two forms of nitrite reductase share recognizable sequence and structural similarity. Synthesis of nitrate assimilation enzymes and uptake systems is controlled by nitrogen limitation in all bacteria examined, but the relevant regulatory proteins exhibit considerable structural and mechanistic diversity in different bacterial groups. A second level of control, pathway-specific induction by nitrate and nitrite in Klebsiella, involves transcription antitermination. Several issues await further experimentation, including the mechanism and energetics of nitrate uptake, the pathway(s) for nitrite uptake, the nature of electron flow during nitrate reduction, and the action of transcriptional regulatory circuits. Fundamental knowledge of nitrate assimilation physiology should also enhance the study of nitrate metabolism in soil, water and other natural environments, a challenging topic of considerable interest and importance.
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PMID:Nitrate assimilation by bacteria. 932 45

Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity. A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity. The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH. This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate. The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components. The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component. The small protein was N-terminally blocked. It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids. The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases. Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp. designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases. Some of the basic properties of both components were determined and their interaction during catalysis was investigated.
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PMID:Two-component flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp. 939 21


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