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)

Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate. The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T). The X-ray analysis of E. coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a novel fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor. Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.
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PMID:The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. 1020 87

In the crystal structure of native p-hydroxybenzoate hydroxylase, Ser212 is within hydrogen bonding distance (2.7 A) of one of the carboxylic oxygens of p-hydroxybenzoate. In this study, we have mutated residue 212 to alanine to study the importance of the serine hydrogen bond to enzyme function. Comparisons between mutant and wild type (WT) enzymes with the natural substrate p-hydroxybenzoate showed that this residue contributes to substrate binding. The dissociation constant for this substrate is 1 order of magnitude higher than that of WT, but the catalytic process is otherwise unchanged. When the alternate substrate, 2,4-dihydroxybenzoate, is used, two products are formed (2,3,4-trihydroxybenzoate and 2,4, 5-trihydroxybenzoate), which demonstrates that this substrate can be bound in two orientations. Kinetic studies provide evidence that the intermediate with a high extinction coefficient previously observed in the oxidative half-reaction of the WT enzyme with this substrate is composed of contributions from both the dienone form of the product and the C4a-hydroxyflavin. During the reduction of the enzyme-2,4-dihydroxybenzoate complex by NADPH with 2, 4-dihydroxybenzoate, a rapid transient increase in flavin absorbance is observed prior to hydride transfer from NADPH to FAD. This is direct evidence for movement of the flavin before reduction occurs.
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PMID:Mechanistic insights into p-hydroxybenzoate hydroxylase from studies of the mutant Ser212Ala. 1032 Mar 59

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhodococcus rhodochrous caused loss of the catalytic activity in a time- and concentration-dependent manner. Peptides (P-81) and (PN-83) were isolated from tryptic digests of the native and tetranitromethane-treated enzyme proteins, respectively. PN-83 was the nitrated form of P-81. The amino acid sequence was GGAPLIDYLESDDDLEFMVYPWPDYFGK (positions 97-124 of the dehydrogenase sequence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less than 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. This indicated that tetranitromethane modifies Y-116 under the experimental conditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid residues, Phe, Ser, or Ala, significantly changed the catalytic activity of the dehydrogenase. All of the mutants contained FAD and exhibited the same spectrophotometric properties as those of the wild type enzyme. The K(m) values for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants changed to large values. The most drastic change was observed for Y116A. The K(d) values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1.5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mutant enzymes exhibited catalytic activities like those of the recombinant enzyme, but the catalytic efficiencies of Y121F and Y121A drastically decreased to 0. 014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104 act on binding of the substrate steroid.
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PMID:Essential tyrosine residues in 3-ketosteroid-delta(1)-dehydrogenase from Rhodococcus rhodochrous. 1050 72

Bacillus subtilis dihydroorotate dehydrogenase (DHOD) consists of two subunits, PyrDI (M(r) = 33,094) and PyrDII (M(r) = 28,099). The two subunits were overexpressed jointly and individually and purified. PyrDI was an FMN-containing flavoprotein with an apparent native molecular mass of 85,000. Overexpressed PyrDII formed inclusion bodies and was purified by refolding and reconstitution. Refolded PyrDII bound 1 mol FAD and 1 mol [2Fe-2S] per mol PyrDII. Coexpression and purification of PyrDI and PyrDII yielded a native holoenzyme complex with an apparent native molecular mass of 114,000 that indicated a heterotetramer (PyrDI(2)PyrDII(2)). The holoenzyme possessed dihydroorotate:NAD(+) oxidoreductase activity and could also reduce menadione and artificial dyes. Purified PyrDI also possessed DHOD activity but could not reduce NAD(+). Compared to PyrDI, the holoenzyme had a more than 20-fold smaller K(m) value for dihydroorotate, an approximately 50-fold smaller K(i) value for orotate, and approximately 500-fold greater catalytic efficiency. Dihydroorotate:NAD(+) oxidoreductase activity could be recovered by mixing the purified subunits. Recovered activity showed a clear dependence on FAD reconstitution of PyrDII but not on reconstitution with FeS clusters. PyrDII had a strong preference for FAD over FMN and bound it with an estimated K(d) value of 4.9 +/- 0.8 nM. pyrDII mutants containing alanine substitutions of the cysteine ligands to the [2Fe-2S] cluster failed to complement the pyr bradytrophy of a DeltapyrDII strain, indicating a requirement for the FeS cluster in PyrDII for normal function in vivo.
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PMID:Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits. 1054 5

Modeling studies of the trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH-ETF) electron transfer complex have suggested potential roles for Val-344 and Tyr-442, found on the surface of TMADH, in electronic coupling between the 4Fe-4S center of TMADH and the FAD of ETF. The importance of these residues in electron transfer, both to ETF and to the artificial electron acceptor, ferricenium (Fc(+)), has been studied by site-directed mutagenesis and stopped-flow spectroscopy. Reduction of the 6-(S)-cysteinyl FMN in TMADH is not affected by mutation of either Tyr-442 or Val-344 to a variety of alternate side chains, although there are modest changes in the rate of internal electron transfer from the 6-(S)-cysteinyl FMN to the 4Fe-4S center. The kinetics of electron transfer from the 4Fe-4S center to Fc(+) are sensitive to mutations at position 344. The introduction of smaller side chains (Ala-344, Cys-344, and Gly-344) leads to enhanced rates of electron transfer, and likely reflects shortened electron transfer "pathways" from the 4Fe-4S center to Fc(+). The introduction of larger side chains (Ile-344 and Tyr-344) reduces substantially the rate of electron transfer to Fc(+). Electron transfer to ETF is not affected, to any large extent, by mutation of Val-344. In contrast, mutation of Tyr-442 to Phe, Leu, Cys, and Gly leads to major reductions in the rate of electron transfer to ETF, but not to Fc(+). The data indicate that electron transfer to Fc(+) is via the shortest pathway from the 4Fe-4S center of TMADH to the surface of the enzyme. Val-344 is located at the end of this pathway at the bottom of a small groove on the surface of TMADH, and Fc(+) can penetrate this groove to facilitate good electronic coupling with the 4Fe-4S center. With ETF as an electron acceptor, the observed rate of electron transfer is substantially reduced on mutation of Tyr-442, but not Val-344. We conclude that the flavin of ETF does not penetrate fully the groove on the surface of TMADH, and that electron transfer from the 4Fe-4S center to ETF may involve a longer pathway involving Tyr-442. Mutation of Tyr-442 likely disrupts electron transfer by perturbing the interaction geometry of TMADH and ETF in the productive electron transfer complex, leading to less efficient coupling between the redox centers.
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PMID:Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein. 1092 12

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.
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PMID:Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase. 1101 18

The kinetics of flavin reduction in two mutant forms of human cytochrome P450 reductase have been studied by stopped-flow spectroscopy with absorption and fluorescence detection. The mutant enzymes were altered at the position of Trp-676, which, by analogy with the structure of rat CPR, is close to the isoalloxazine ring of the enzyme-bound FAD. We show that mutant CPRs in which Trp-676 has been changed to histidine (W676H) and alanine (W676A) can be reduced by NADPH only to the two-electron level in single mixing stopped-flow experiments. The concentration dependence of the rate of hydride transfer indicates that the second, noncatalytic NADPH-binding site present in wild-type CPR is retained in the mutant enzymes. Detailed studies of W676H CPR indicate that further reduction of the enzyme beyond the two electron level is prevented due to the slow release of NADP(+) from the active site following the first hydride transfer from NADPH, owing to the stability of a reduced enzyme-NADP(+) charge-transfer complex. Reduction to the four-electron level is achieved in a sequential mixing stopped-flow experiment. In this procedure, W676H CPR is reacted first with a stoichiometric amount of NADPH, and then, following a delay of 100 ms, with excess NADPH. The data indicate that occupancy of the noncatalytic coenzyme site also hinders NADP(+) release from reduced enzyme. Fluorescence stopped-flow studies of the W676H and wild-type CPR enzymes reveal that the complex signals associated with reduction of wild-type CPR by NADPH are attributable to changes in the environment of residue W676. From these studies, a model is proposed for nicotinamide binding in wild-type CPR. In this model W676 serves as a trigger to release NADP(+) from the active site following hydride transfer. In the W676H enzyme, the slow release of NADP(+) is a consequence of the combined effects of (i) removing W676 by mutagenesis (thus removing the trigger for displacement) and (ii) the binding of NADPH in the noncatalytic site, thus trapping NADP(+) in the catalytic site.
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PMID:Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases. 1112 26

A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent k(cat) of W676A is equivalent (90%) to the NADPH-dependent k(cat) of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent K(M)(NADPH) and K(M)(NADH) values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2'-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system.
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PMID:Engineering of a functional human NADH-dependent cytochrome P450 system. 1113 48

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.
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PMID:Purification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei strain KUFI-6N. 1121 Jan 39

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.
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PMID:alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone. 1128 59


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