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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The characteristic red color of some photosynthetic bacteria and the orange color of Neurospora conidia is due to the presence of carotenoids, photoprotective pigments synthesized by plants, algae, bacteria, and fungi. Generally, carotenoids are tetraterpenes in which absorption of visible light and photoprotection are mediated by a chain of conjugated double bonds, the chromophore, which is formed by successive desaturations of phytoene, a colorless precursor. The genes al-1 and crtI mediate the desaturation of phytoene in Neurospora crassa and Rhodobacter capsulatus, respectively. Here, we report that alignment of the primary sequence of Al-1, CrtI, and CrtD, another carotenoid desaturase, reveals conservation with amino acid residues that mediate FAD-binding and dimerization functions in Azotobacter vinelandii dihydrolipoamide dehydrogenase and human glutathione reductase, two disulfide oxidoreductases. Plasmids containing the coding region of an al-1 cDNA fused to appropriate bacterial transcriptional and translational signals complement crtI mutants. Our results indicate that both structure and function of carotenoid desaturases have been conserved during evolution and suggest that these enzymes are evolutionarily related to disulfide oxidoreductases.
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PMID:Carotenoid desaturases from Rhodobacter capsulatus and Neurospora crassa are structurally and functionally conserved and contain domains homologous to flavoprotein disulfide oxidoreductases. 214 93

Alcohol oxidase (AO) expressed in transformed oleic acid-grown Saccharomyces cerevisiae, accumulated into microbodies to up to 8% of the total protein content of the organelles. This led to a small increase in volume fraction of the organelles, but not in their number. Most of the AO protein was present in large aggregates in the cytosol. The AO synthesized was inactive, irrespective of its subcellular localization and did not contain FAD. When the same AO gene was expressed in fused protoplasts of transformed S. cerevisiae and Hansenula polymorpha, the enzyme was properly assembled and activated in H. polymorpha microbodies.
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PMID:Heterologous expression of alcohol oxidase in Saccharomyces cerevisiae: properties of the enzyme and implications for microbody development. 265 78

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

The precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase from pea was expressed in Escherichia coli as a carboxyl-terminal fusion to glutathione S-transferase. The fused protein was soluble, and the precursor could be purified in a few steps involving affinity chromatography on glutathione-agarose, cleavage of the transferase portion by protease Xa, and ion exchange chromatography on DEAE-cellulose. The purified prereductase contained bound FAD but displayed marginally low levels of activity. Removal of the transit peptide by limited proteolysis rendered a functional protease-resistant core exhibiting enzymatic activity. The FAD-containing precursor expressed in E. coli was readily transported into isolated pea chloroplasts and was processed to the mature size, both inside the plastid and by incubation with stromal extracts in a plastid-free reaction. Import was dependent on the presence of ATP and was stimulated severalfold by the addition of plant leaf extracts.
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PMID:The precursor of pea ferredoxin-NADP+ reductase synthesized in Escherichia coli contains bound FAD and is transported into chloroplasts. 765 8

We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.
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PMID:Folding, flavinylation, and mitochondrial import of 6-hydroxy-D-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase. 771 2

Monodehydroascorbate radicals are generated in plant cells enzymatically by the hydrogen peroxide scavenging enzyme, ascorbate peroxidase, and nonenzymatically via the univalent oxidation of ascorbate by superoxide, hydroxyl, and various organic radicals. Regeneration of ascorbate is achieved by monodehydroascorbate reductase (EC 1.6.5.4) using NAD(P)H as an electron donor or, alternatively, by a set of two coupled reactions requiring dehydroascorbate reductase, glutathione reductase, glutathione, and NAD(P)H. As monodehydroascorbate reductase is a key enzyme in maintaining reduced pools of ascorbate, an important antioxidant, we undertook this study to learn more about its structure, function, and regulation. Herein we report the molecular cloning and characterization of a cDNA encoding monodehydroascorbate reductase of pea (Pisum sativum L.). The cDNA encodes a 433-amino acid polypeptide that shows, respectively, 73 and 87% identity with peptide fragments from soybean and cucumber monodehydroascorbate reductase. Monodehydroascorbate reductase contains the NAD(P)H and FAD binding domains of other flavin oxidoreductases. The cloned enzyme lacks a transit peptide, but the sequence of the carboxyl terminus is Ser-Lys-Ile, similar to the targeting motif found in peroxisomal proteins. When expressed in Escherichia coli fused to maltose-binding protein, monodehydroascorbate reductase has enzymatic properties comparable with purified soybean and cucumber monodehydroascorbate reductase. Northern blot analysis shows that the monodehydroascorbate reductase transcript is 1.6 kilobase in size and is expressed at relatively low levels in all plant tissues examined.
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PMID:Molecular cloning and characterization of a cDNA encoding pea monodehydroascorbate reductase. 798 54

Fatty acid subterminal (omega-1 approximately omega-3) hydroxylase of the fungus Fusarium oxysporum was solubilized from the microsomal fraction and partially purified. The hydroxylase activity was recovered into a single active fraction, and its spectral nature showed the presence of cytochrome P-450 (P-450). Fatty acid hydroxylase activity was markedly restored upon addition of FAD, FMN, and/or hemin to the eluted fraction. The fraction also exhibited other properties characteristic of both a hemeprotein and a flavin-containing reductase. These results are highly indicative that the fungal hydroxylase is a fused protein containing both P-450 and its reductase domains. In this aspect the fungal enzyme resembles bacterial P-450BM3, although it is membrane-bound unlike the bacterial counterpart.
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PMID:Fatty acid hydroxylase of the fungus Fusarium oxysporum is possibly a fused protein of cytochrome P-450 and its reductase. 803 65

Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells. The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C. The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively. The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme. The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1. These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product. On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively. The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase. 816 54

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.
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PMID:One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase. 828 51

The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule.
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PMID:Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution. 855 26


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