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 hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.
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PMID:Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC. 0 Dec 80

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.
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PMID:Immobilization and characterization of D-amino acid oxidase. 3 57

Point mutations in the gene of pyruvate oxidase from Lactobacillus plantarum, with proline residue 178 changed to serine, serine 188 to asparagine, and alanine 458 to valine, as well as a combination of the three single point mutations, lead to a significant functional stabilization of the protein. The enzyme is a tetrameric flavoprotein with tightly bound cofactors, FAD, TPP, and divalent metal ions. Thus, stabilization may be achieved either at the level of tertiary or quaternary interactions, or by enhanced cofactor binding. In order to discriminate between these alternatives, unfolding, dissociation, and cofactor binding of the mutant proteins were analyzed. The point mutations do not affect the secondary and tertiary structure, as determined by circular dichroism and protein fluorescence. Similarly, the amino acid substitutions neither modulate the enzymatic properties of the mutant proteins nor do they stabilize the structural stability of the apoenzymes. This holds true for both the local and the global structure with unfolding transitions around 2.5 M and 5 M urea, respectively. On the other hand, deactivation of the holoenzyme (by urea or temperature) is significantly decreased. The most important stabilizing effect is caused by the Ala-Val exchange in the C-terminal domain of the molecule. Its contribution is close to the value observed for the triple mutant, which exhibits maximum stability, with a shift in the thermal transition of ca. 10 degrees C. The effects of the point mutations on FAD binding and subunit association are interconnected. Because FAD binding is linked to oligomerization, the stability of the mutant apoenzyme-FAD complexes is increased. Accordingly, mutants with maximum apparent FAD binding exhibit maximum stability. Analysis of the quaternary structure of the mutant enzymes in the absence and in the presence of coenzymes gives clear evidence that both improved ligand binding and subunit interactions contribute to the observed thermal stabilization.
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PMID:Characterization of the stabilizing effect of point mutations of pyruvate oxidase from Lactobacillus plantarum: protection of the native state by modulating coenzyme binding and subunit interaction. 130

D-Amino acid oxidase purified from the yeast Rhodotorula gracilis is a flavoenzyme which does not require exogenous FAD for maximum activity. The enzyme showed temperature and pH activity optima centred between 40 and 45 degrees C and between 8.0 and 8.5, respectively; a broad pH and ionic strength range of stability and a more limited range of thermostability was determined. The enzyme stability was markedly influenced by the presence of 2-mercaptoethanol. Apparent kinetic parameters for a number of substrates were determined: nonpolar and aromatic D-amino acids appeared to be the best substrates. Steady state measurements carried out at different oxygen concentrations indicated that for D-alanine the kinetic pattern is consistent with a Ping Pong Bi Bi mechanism; kcat values on D-alanine and D-valine are 43,250 min-1 and 31,370 min-1, respectively. L-Amino acids did not inhibit enzyme activity; several aromatic and aliphatic carboxylic acids proved to be competitive inhibitors of the enzyme and their ki values were determined. The reported properties of R. gracilis D-amino acid oxidase markedly distinguish it from other characterized D-amino acid oxidases.
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PMID:Specificity and kinetics of Rhodotorula gracilis D-amino acid oxidase. 134 88

We previously reported the expression of a full-length cDNA complementary to a rat liver NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) mRNA in Escherichia coli (Q. Ma, R. Wang, C. S. Yang, and A. Y. H. Lu, 1990, Arch. Biochem. Biophys. 283, 311-317). Since cysteine residues have been suggested to be important for the catalysis of flavoproteins and a lysine residue at position 76 in NAD(P)H:quinone oxidoreductase has been proposed to be involved in electron transfer of the enzyme, we investigated the roles of lysine 76 and cysteine 179 of this enzyme in catalysis by site-directed mutagenesis. Mutant cDNA clones replacing lysine 76 with valine (K76V) and cysteine 179 with alanine (C179A) were generated by a procedure based on the polymerase chain reaction. The mutant enzymes were expressed in E. coli. The cytosolic activities of the K76V and C179A mutants were 50 and 25% of that of the wild type (DTD), due to lower levels of the mutant proteins as shown by immunoblot analysis. The mutant proteins were purified to apparent homogeneity. The purified K76V and C179A mutant enzymes maintained full activities of 2,6-dichlorophenolindophenol (DCIP) reduction compared with that of the wild type. The mutant enzymes exhibited kinetic parameters for DCIP, NADH, and NADPH similar to those of DTD except that, with K76V, the Km for NADPH was doubled. Both mutant proteins contained two molecules of FAD per enzyme molecule. Dicumarol inhibited K76V and C179A mutant activities to greater than 90% at a concentration of 10(-7) M. Heat stability studies showed that C179A was much more sensitive to inactivation at 37 degrees C than both the wild-type and K76V enzymes. It is concluded from this study that lysine 76 and cysteine 179 are not essential in catalysis and in the binding of FAD, DCIP, and dicumarol. However, lysine residue 76 appears to play a role in NADPH binding and cysteine residue 179 is important in maintaining the stability of the enzyme.
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PMID:Site-directed mutagenesis of rat liver NAD(P)H: quinone oxidoreductase: roles of lysine 76 and cysteine 179. 156 99

The gene encoding the valine-resistant and FAD-independent acetolactate synthase of Klebsiella pneumoniae was isolated and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and it exhibited an open reading frame of 1680 bp in length. In vivo expression of the acetolactate synthase-encoding gene in E. coli revealed a single 60-kDa protein which is consistent with the molecular weight calculated from the deduced amino acid sequence of the gene product. The gene product shares about 20-30% homology with the acetolactate synthases of E. coli, yeast and higher plants.
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PMID:Cloning, sequencing and heterologous expression of a Klebsiella pneumoniae gene encoding an FAD-independent acetolactate synthase. 164 3

Flavin-containing monooxygenase (FMO; EC 1.14.13.8) was purified from mouse kidney microsomes and compared to that isolated from mouse liver microsomes. The purified enzymes from kidney and liver appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 58,000 daltons. On wide range (pH 3.5 to 9.0) isoelectric focusing, FMOs from kidney and liver resolved as a single band with an isoelectric point of 8.2. The enzymes from both kidney and liver have a pH optimum of 9.2. Thiobenzamide-S-oxidation catalyzed by both enzymes was sensitive to inhibition by the competitive inhibitors thiourea and methimazole. At an n-octylamine concentration of 3 mM, thiobenzamide-S-oxidation by the kidney FMO was increased by 122% and that by the liver FMO by 148%. Km and Vmax values were determined and compared between the two tissue enzymes for xenobiotic substrates containing nucleophilic nitrogen, sulfur or phosphorus atoms. In general, for most FMO substrates, Km and Vmax values were similar between kidney and liver FMO with only a few exceptions. The Km and Vmax values for fenthion for kidney were only half of those observed for liver FMO. Fonofos was unusual in having a low Km as well as a low Vmax for both tissue enzymes. Anti-sera developed to the FMO purified from kidney and liver showed cross-reactivity with each purified enzyme as well as with a protein with the same molecular weight as the purified FMO present in both kidney and liver microsomes. These bands showed equal intensity based on an equivalent amount of protein. Analysis of kidney and liver FMO by proteolytic digestion followed by visualization of peptides by silver staining or immunoblotting showed only minor differences between the enzymes of the two tissues. The amino acid composition of both mouse kidney and liver FMO was low in methionine and histidine and rich in aspartate/asparagine, glutamate/glutamine, leucine, valine and glycine. Edman degradation of the purified mouse kidney and liver FMO provided a single amino acid sequence of the NH2-terminus. This sequence matched exactly with the cDNA-deduced sequence reported for the pig and rabbit liver beginning with the fifth amino acid and contained the highly conserved FAD-binding domain Gly-X-Gly-X-X-Gly, commonly found in a number of other FAD-binding proteins. These studies indicate that the renal and hepatic forms of FMO from mouse are similar enzymes that are immunologically related and show only a few minor differences.
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PMID:The flavin-containing monooxygenase of mouse kidney. A comparison with the liver enzyme. 193 Feb 64

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and greater than 1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.
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PMID:Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp. 365 79

A reproducible scheme has been developed for the preparation of rat liver thioredoxin and thioredoxin reductase (EC 1.6.4.5) by using assays based on reduction of insulin and 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Both proteins were purified to homogeneity, as judged from polyacrylamide gel electrophoresis. Thioredoxin had a molecular weight of 12 000 and contained about 110 amino acids including 4 half-cystines and an NH2-terminal valine. Peptide maps of reduced and carboxymethylated thioredoxin showed that the protein had the active center sequence -Cys-Gly-Pro-Cys-Lys-Met- characteristic of thioredoxins also from procaryotes. Prolonged air oxidation of fully reduced thioredoxin created inactive, aggregated disulfide-containing molecules. Thioredoxin reductase showed a subunit molecular weight of 58 000 and a native molecular weight of 116 000. The enzyme was highly specific for NADPH with a Km of 6 microM. It contained FAD as prosthetic group and was sensitive to inhibition by arsenite. Thioredoxin reductase had a Km of 2.5 microM for rat and calf liver thioredoxin and a Kcat of 3000 min-1.
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PMID:Rat liver thioredoxin and thioredoxin reductase: purification and characterization. 715 51

Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.
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PMID:Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida. 720 73


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