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 flavoenzymes dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) contain covalently bound FAD linked via the 8 alpha-position of the isoalloxazine ring to the imidazole N(3) of a histidine residue (Cook, R. J., Misono, K. S., and Wagner, C. (1984) J. Biol. Chem. 259, 12475-12480). The flavin-peptides from tryptic digests of these two enzymes have been isolated and sequenced. Automated sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase contained 25 amino acid residues in the following sequence: Ser-Glu-Leu-Thr-Ala-Gly-Ser- Thr-Trp-His(flavin)-Ala-Ala-Gly-Leu-Thr-Thr-Tyr-Phe-His-Pro-Gly-Ile-A sn-Leu-Lys. The sequence determined for the flavin-peptide from sarcosine dehydrogenase contained 14 amino acid residues Leu-Thr-Ser-Gly-Thr-Thr-Trp-His(flavin)-Thr-Ala-Gly-Leu-Gly-Arg.
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PMID:The amino acid sequences of the flavin-peptides of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 405 29

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.
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PMID:Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase. 613 4

Very small quantities of FAD were able to reactivate apo-D-amino acid oxidase. In the presence of D-alanine, luminol, horseradish peroxidase, and an excess of the apoenzyme, a quantitative luminometric determination of FAD was possible. The maximal photon emission measured in a bicarbonate buffer, pH 9.2, at 37 degrees C was proportional to the amount of FAD added. FMN, riboflavin, or 5-deazaflavin produced no chemiluminescence and had no inhibitory effect in the assay when added together with FAD. With this method, FAD could be quantitatively determined with high accuracy in perchloric acid extracts of animal tissue and bacteria.
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PMID:Luminometric determination of FAD in subpicomole quantities. 613 73

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study. 614 80

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are the folate binding proteins of rat liver mitochondria. These two enzymes contain covalently bound flavin and catalyze similar oxidative demethylation reactions (Wittwer, A. J., and Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108). Flavin-peptides have been purified from these two enzymes after proteolytic digestion by trypsin and chymotrypsin. The spectral and chromatographic properties of these flavin peptides changed after treatment with nucleotide pyrophosphatase in a manner consistent with the conversion of an FAD-peptide to an FMN-peptide. The pKa for pH-dependent fluorescence quenching of the purified flavin-peptides was not affected by borohydride reduction which, in conjunction with the pKa values, indicated that the flavin was covalently linked via the 8 alpha position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Peptides from both enzymes showed histidylflavin at the N terminus. Amino acid composition and sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase was His(flavin)-Ala-Ala-Gly-Leu. Amino acid composition and N-terminal analysis suggested the sequence of the flavin-peptide of sarcosine dehydrogenase was His(flavin)-(Ala, Gly,Thr)-Leu.
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PMID:Identification of the covalently bound flavin of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 649 Jun 27

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.
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PMID:Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa. 681 40

Cocaine is reported to produce either periportal or mid-zonal necrosis in mice pretreated with the enzyme inducer phenobarbitone (James et al. 1987; Powell et al. 1991; Charles & Powell 1992). Dose-response and time course experiments were performed in phenobarbitone treated male DBA/2Ha mice to study the pathogenesis of this unusual cocaine induced lesion. An increase in the dose of cocaine from 60 to 90 or 120 mg/kg produced more extensive and severe periportal and linking portal damage and elevated plasma aspartate (AST) and alanine (ALT) aminotransferases in a dose dependent manner. Scattered hepatocyte degeneration began at the edge of the periportal region and was detectable by electron microscopy within 30 minutes of administration of 60 mg/kg of cocaine, with conspicuous disorganization of the endoplasmic reticulum being one of the earliest changes. Significant elevations of plasma AST and ALT were observed 3 hours after cocaine administration and were sustained for 12 hours, at which time progressive hepatocyte damage had developed into a network of confluent necrosis at the periphery of the periportal region. The rapidity of organelle derangement and subsequent cell death, and absence of any effect on total cytochrome P-450 or FAD-mono-oxygenase levels, appear to distinguish this periportal lesion from previous reports of cocaine induced centrilobular necrosis in non-enzyme induced mice, suggesting that the two types of damage may develop by different mechanisms. The observation that periportal lesions commence at the periphery of the periportal area, progressing portalwards with increasing dose and time, offers an explanation for the previously conflicting reports of cocaine induced mid-zonal and/or periportal lesions in phenobarbitone treated mice.
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PMID:Cocaine hepatotoxicity: a study on the pathogenesis of periportal necrosis. 773 31

The gene for trypanothione reductase from the Silvio strain of Trypanosoma cruzi has been cloned, sequenced and overexpressed in Escherichia coli using the constitutive lpp promoter on the expression plasmid pBSTNAV. Up to 13% of the total soluble protein is enzymically active trypanothione reductase with kinetic properties similar to the enzyme purified from T. cruzi. In order to assess the catalytic role of the putative active-site cysteine residues (C53 and C58), three mutant proteins have been constructed by site-directed mutagenesis substituting alanine or serine residues for cysteine; [C53A]trypanothione reductase, [C53S]trypanothione reductase and [C58S]trypanothione reductase. Although the purified, recombinant mutant proteins were catalytically inactive with NADPH and trypanothione disulphide as substrates, all showed comparable levels of transhydrogenase activity between NADPH and thio-NADP+, suggesting that the mutant proteins had correctly folded in vivo. All three mutants showed substantially different catalytic parameters for thio-NADP+ than the wild-type enzyme, presumably as a consequence of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity. The purified [C58S]trypanothione reductase showed spectral properties similar to the oxidised wild-type enzyme but, unlike the wild-type enzyme, did not acquire the characteristic charge-transfer complex of the EH2 form on addition of NADPH. In contrast, in the absence of NADPH both [C53A]trypanothione reductase and [C53S]trypanothione reductase showed spectral properties similar to the EH2 form of the wild-type enzyme. These data indicate that both C53 and C58 are essential for overall catalysis, with the thiolate anion of C58 interacting with the enzyme-bound FAD and C53 interacting with the disulphide substrate. These mutants should be useful in crystallographic studies of reaction intermediates which cannot be obtained with the catalytically active native enzyme.
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PMID:Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase. 773 73

The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.
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PMID:Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger. 777 50

The flavoprotein NADH peroxidase from Enterococcus faecalis 10C1 has been shown to contain, in addition to FAD, an unusual cysteine-sulfenic acid (Cys-SOH) redox center. The non-flavin center cycles between reduced (Cys-SH) and oxidized (Cys-SOH) states, and the 2.16 A crystal structure of the non-native cysteine-sulfonic acid (Cys-SO3H) form of the wild-type peroxidase supports the proposed catalytic role of Cys42. In this study, we have employed a site-directed mutagenesis approach in which Cys42 is replaced with Ser and Ala, neither side chain of which is capable of redox activity. Reductive titrations of both C42S and C42A mutants lead directly to full FAD reduction with 1 equiv of either dithionite or NADH, consistent with elimination of the Cys-SOH center. Direct determinations of the redox potentials for the FAD/FADH2 couples yield values of -219 and -197 mV, respectively, for C42S and C42A peroxidases, indicating that the presence of Cys42-SH in the two-electron-reduced wild-type enzyme lowers the flavin potential by approximately 100 mV. Anaerobic stopped-flow analyses of the reduction of C42S and C42A peroxidases by NADH demonstrate that in both cases flavin reduction is rapid; these results are confirmed by enzyme-monitored, steady-state kinetic analyses which, in addition, give turnover numbers approximately 0.04% that of wild-type enzyme. These results are entirely consistent with the role proposed for Cys42 in the catalytic redox cycle of wild-type NADH peroxidase and indirectly support its function as a peroxidatic center in the homologous NADH oxidase.
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PMID:Analysis of the kinetic and redox properties of NADH peroxidase C42S and C42A mutants lacking the cysteine-sulfenic acid redox center. 781 35


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