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 major soluble cytochrome isolated from microaerobically grown cells of Shewanella putrefaciens has been shown to be a novel type of flavocytochrome with fumarate reductase activity. This flavocytochrome, located in the periplasmic fraction of cell extracts, has been purified to homogeneity and shown to contain 4 mol of haem c and 1 mol of non-covalently bound FAD per mol of protein. An M(r) value of 63,800 is estimated from sequence analysis assuming 4 mol of haem/mol of protein. In the presence of the artificial electron donor, reduced methyl viologen, the flavocytochrome catalysed the reduction of fumarate but not that of nitrite, dimethylsulphoxide, trimethylamine-N-oxide or sulphite. The pH optimum was 7.4 with calculated pKa values of 6.8 and 8.0 for contributing catalytic groups. The Km and kcat. values for fumarate reduction were 21 microM and 250 s-1 respectively, whereas the corresponding values for succinate oxidation with 2,6-dichlorophenol-indophenol as electron carriers were 200 microM and 0.07 s-1 respectively. Mesaconic acid was a competitive inhibitor of fumarate reduction with a Ki of 2 microM. Zymogram staining of polyacrylamide gels with purified protein showed a band of fumarate reductase activity. Polyclonal antibodies, raised to the purified flavocytochrome, were shown to titrate out fumarate reductase activity. We conclude that the physiological role of this enzyme is as a fumarate reductase. Optical absorption spectra of the flavocytochrome indicated that all the haems were of the c-type and gave alpha, beta and gamma peaks at 552.3, 523 and 418 nm in the reduced spectrum with epsilon values of 30.2, 15.9 and 188.2 mM-1.cm-1 respectively. Oxidized spectra showed no 695 nm band that would be indicative of His-Met coordination. Two redox potentials were resolved at -220 mV and -320 mV. The cytochrome was reduced by formate in the presence of particulate cell fractions. The relationship of this cytochrome to other low-potential flavocytochromes c is discussed.
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PMID:Purification and properties of a novel cytochrome: flavocytochrome c from Shewanella putrefaciens. 809 12

Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH. 821 22

1. Isolated colonic epithelial cells of the rat were incubated for 40 min with [6-14C]glucose and n-[1-14C]butyrate in the presence of 0.1-2.0 mmol/l NaHS, a concentration range found in the human colon. Metabolic products, 14CO2, acetoacetate, beta-hydroxybutyrate and lactate, were measured and injury to cells was judged by diminished production of metabolites. 2. Oxidation of n-butyrate to CO2 and acetoacetate was reduced at 0.1 and 0.5 mmol/l NaHS, whereas glucose oxidation remained unimpaired. At 1.0-2.0 mmol/l NaHS, n-butyrate and glucose oxidation were dose-dependently reduced at the same rate. 3. To bypass short-chain acyl-CoA dehydrogenase activity necessary for butyrate oxidation, ketogenesis from crotonate was measured in the presence of 1.0 mmol/l NaHS. Suppression by sulphide of ketogenesis from crotonate (-10.5 +/- 6.1%) compared with control conditions was not significant, whereas suppression of ketogenesis from n-butyrate (-36.00 +/- 5.14%) was significant (P = < 0.01). Inhibition of FAD-linked oxidation was more affected by NaHS than was NAD-linked oxidation. 4. L-Methionine (5.0 mmol/l) significantly redressed the impaired beta-oxidation induced by NaHS. Methionine equally improved CO2 and ketone body production, suggesting a global reversal of the action of sulphide. 5. Sulphide-induced oxidative changes closely mirror the impairment of beta-oxidation observed in colonocytes of patients with ulcerative colitis. A hypothesis for the disease process of ulcerative colitis is that sulphides may form persulphides with butyryl-CoA, which would inhibit cellular short-chain acyl-CoA deHydrogenase and beta-oxidation to induce an energy-deficiency state in colonocytes and mucosal inflammation.
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PMID:Sulphide impairment of substrate oxidation in rat colonocytes: a biochemical basis for ulcerative colitis? 828 51

The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme.
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PMID:Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate. 835 49

The structural features of the active site of human monoamine oxidase B (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl]-5-chloro-2-pyridine carboxamide HCl (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH(3)CN. Analysis of the flavin spectrum of [(3)H]lazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that [(3)H]lazabemide is incorporated into the MAO-B peptide stretch containing the FAD-modified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that mutation of Cys389 with a Ser residue does not markedly affect the activity of the enzyme [Wu, H.-F., Chen, K. and Shih, J.C. (1993) Mol. Pharmacol. 43, 888-893] the mutant carrying an Ala at this position was virtually inactive. Conversely, substitution of Lys386 (to Met) and Ser394 (to Ala) did not markedly modify the kinetic properties of the enzyme. We also report that mutation of MAO-B Thr158 (to Ala) resulted in a dramatic loss of enzymic activity.
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PMID:Investigation on the structure of the active site of monoamine oxidase-B by affinity labeling with the selective inhibitor lazabemide and by site-directed mutagenesis. 866 24

A gene coding for the salicylate hydroxylase has been isolated from chromosomal DNA of Pseudomonas putida S-1 and sequenced. The DNA fragment contained an open reading frame of 1266 bp encoding a polypeptide of 421 amino acid residues. The predicted amino acid sequence of the protein gave a good agreement with the sequences determined with the peptides isolated from the enzyme but methionine residue in the amino terminal was deleted in the N-terminal sequence of the enzyme protein. The nucleotide and amino acid sequences of the salicylate hydroxylase shared several common characteristics with those of the enzyme encoded on the plasmid DNA of P. putida PpG7; homology of nucleotide sequence is 58% and that of amino acid sequence is 56%. We could find two large conserved regions of the amino acid sequence at or near FAD- and NADH-binding regions. The FAD-binding site locates on the amino terminal and a lysine residue, functioning as an NADH-binding site (K. Suzuki, M. Mizuguchi, T. Gomi, and E. Itagaki, 1995, J. Biochem. 117,579-585), locates as Lys163.
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PMID:Structure of chromosomal DNA coding for Pseudomonas putida S-1 salicylate hydroxylase. 869 32

Thioredoxin exists in all organisms and is responsible for the hydrogen transfer to important enzymes for ribonucleotide reduction and the reduction of methionine sulphoxide and sulphate. Thioredoxins have also been shown to regulate enzyme activity in plants and are also involved in the regulation of transcription factors and several other regulatory activities. Thioredoxin is reduced by the flavoenzyme thioredoxin reductase using NADPH. We have now determined the first structure of a eukaryotic thioredoxin reductase, from the plant Arabidopsis thaliana, at 2.5 A resolution. The dimeric A. thaliana thioredoxin reductase is structurally similar to that of the Escherichia coli enzyme, and most differences occur in the loops. Because the plant and E. coli enzymes have the same architecture, with the same dimeric structure and the same position of the redox active disulphide bond, a similar mechanism that involves very large domain rotations is likely for the two enzymes. The subunit is divided into two domains, one that binds FAD and one that binds NADPH. The relative positions of the domains in A. thaliana thioredoxin reductase differ from those of the E. coli reductase. When the FAD domains are superimposed, the NADPH domain of A. thaliana thioredoxin reductase must be rotated by 8 degrees to superimpose on the corresponding domain of the E. coli enzyme. The domain rotation we now observe is much smaller than necessary for the thioredoxin reduction cycle.
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PMID:Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution. 900 Jun 29

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.
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PMID:alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein. 910 14

The fadH gene coding for an NADPH-dependent 2.4-dienoyl-CoA reductase from Escherichia coli has been cloned by the polymerase chain reaction. This gene is located at 67.65 min on the E. coli chromosome. The complete open reading frame contains 2019 bp coding for the processed protein of 671 amino acid residues, with a calculated molecular mass of 72.55 kDa, which lacks the N-terminal methionine. Construction and expression of the plasmid pNDH, which contained the fadH gene under the control of the T7 promoter, resulted in a 110-fold increase in the reductase activity above the level detected in E. coli cells containing the control vector. The kinetic parameters of the purified reductase were determined to be 50 microM and 2.3 microM for the Km values of NADPH and 2-trans, 4-trans-decadienoyl-CoA, respectively, and 16 s(-1) for the k(cat) value. Analysis of the kinetic data revealed that the reaction catalyzed by this enzyme proceeds via a ping-pong mechanism. The observed dissimilarity between the E. coli and mammalian 2,4-dienoyl-CoA reductase sequences suggests that they have evolved from distinct ancestral genes. Sequence analysis also suggests that the N-terminal part of the E. coli reductase contains the FAD-binding domain whereas the NADPH-binding domain is located in the C-terminal region of the protein.
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PMID:Cloning and expression of the fadH gene and characterization of the gene product 2,4-dienoyl coenzyme A reductase from Escherichia coli. 934 10

The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacterium Synechocystis sp. strain PCC 6803. In the present work the gene (lpdA; database accession number Z48564) encoding the apoprotein of this LPD in Synechocystis PCC 6803 has been identified, sequenced and analysed. The lpdA gene codes for a protein starting with methionine, which is post-translationally removed. The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD). The LPD is an acidic protein with a calculated isoelectric point of 5.17. Comparison of the amino acid sequence of the Synechocystis LPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published. As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-free Synechocystis mutant by insertionally inactivating the lpdA gene with a kanamycin-resistance cassette. However, the selected transformants appeared to be heteroallelic, containing both the intact lpdA gene and the lpdA gene inactivated by the drug-resistance cassette. The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium. Growth over a prolonged time was only possible after an increased buffering of the medium. Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.
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PMID:Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp. strain PCC 6803. 938 33


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