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)

To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae. All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate. The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes. On purification and further characterization, these two mutants were found to each contain covalent FAD. Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A. Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin. In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme. Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05. The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77. These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues. The mechanism of C-H abstraction is therefore unaltered. The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.
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PMID:Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A. 1173 3

Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.
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PMID:Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A. 1175 41

The high-level expression, purification, and characterization of recombinant membrane-bound human liver monoamine oxidase A (MAO-A) in Pichia pastoris is described. Two liters of fermentation culture produces 1170 units (660 mg) of MAO-A. The enzyme is purified in a 35% yield, is homogeneous on denaturing gel electrophoresis, and exhibits a single species (60,512 +/- 6 Da) on electrospray mass spectrometry. It contains 1 mol of 8alpha-S-cysteinyl FAD/mole of enzyme and exhibits >95% functionality. In contrast, the Saccharomyces cerevisiae-expressed enzyme is partially processed by C-terminal serine removal as demonstrated by mass spectra. The amino termini of both P. pastoris- and S. cerevisiae-expressed MAO-A are acetylated on the N-terminal methionine. The steady-state kinetic properties of P. pastoris-expressed MAO-A are similar to those of S. cerevisiae-expressed MAO-A using the following substrates: phenethylamine, p-CF(3)-benzylamine, dopamine, serotonin, and kynuramine. Reductive titrations demonstrate that the recombinant enzyme is reduced by 1 mol of substrate or dithionite as expected for the two electron equivalents required for flavin reduction. Absorption and EPR spectra show no radical species in the resting enzyme while the anionic flavin radical is formed in 50% yield during the reductive titration with dithionite. These data demonstrate significant advantages in the heterologous expression of human MAO-A in P. pastoris compared with the published S. cerevisiae system in higher expression level (329 mg/L) and in a higher level of homogeneity of the isolated enzyme.
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PMID:High-level expression of human liver monoamine oxidase A in Pichia pastoris: comparison with the enzyme expressed in Saccharomyces cerevisiae. 1181 36

Reversible inhibitors of monoamine oxidase A (MAO A) are used as antidepressants. The influence of inhibitors such as pirlindole (pyrazinocarbazole) on the redox co-factor (flavin adenine dinucleotide, FAD) is a key factor in the inhibition. The kinetic, spectral, and thermodynamic changes induced by four closely related pirlindole analogues have been determined to investigate their interaction with the FAD in the active site of MAO A. For a model of flavin-inhibitor stacking, more favourable association would be expected between lumiflavin and the flatter analogues with a double bond at N3, and indeed lower K(i) values were found. However, the spectral changes induced by inhibitor binding to MAO A were 45% less for inhibitors with a double bond. Both in the absence and presence of the double bond, compounds with cyclohexyl at C8 induced 85% larger decrease in absorbance at 500nm than did those with a methyl substituent. In contrast, the K(i) values for the cyclohexyl compounds were lower, indicating greater affinity despite the lower perturbation of the flavin spectrum. All inhibitors stabilised the semiquinone of the FAD when MAO A was titrated with dithionite and prevented further reduction. These results indicate that the active site of MAO A is far more sensitive to structural variation than would be predicted by the simple flavin stacking model. Further, the independent changes in inhibitory potency and flavin perturbation preclude direct interaction with the flavin as a mode of binding and indicate that inhibitor-protein interactions must be important for inhibition.
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PMID:Monoamine oxidase A inhibitory potency and flavin perturbation are influenced by different aspects of pirlindole inhibitor structure. 1278 38

The structural details of the interactions of the covalent 8alpha-S-cysteinyl-FAD with the protein moiety in monoamine oxidase B (MAO B) based on the MAO B crystal structure are described. The dinucleotide is bound to the protein in an extended conformation with the majority of the bonds to the protein identified as hydrogen bonds with amino acid side chains, amide bonds, and water molecules. Since those amino acids interacting with the FAD are conserved in monoamine oxidase A (MAO A), it is proposed that the FAD binding site in MAO A is quite similar to that in MAO B. The redox-active isoalloxazine ring is buried in the protein without direct access to bulk solvent. An electrostatic interaction is observed between the anionic pyrophosphate moiety and Arg42. The normally flat oxidized flavin ring is in a bent, puckered conformation in the MAO B binding site which is suggested to contribute to its reactivity in catalysis. This structural information is then used to explain previous studies on flavin analog incorporation into either MAO B or into MAO A.
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PMID:The FAD binding sites of human monoamine oxidases A and B. 1469 81

Reversible monoamine oxidase A inhibitors (RIMA) are used as antidepressants but little is known about how they interact with the active site of the enzyme. Heterologous expression of human liver MAO-A in yeast provides sufficient protein for molecular studies and direct observation of the changes in the spectrum of the FAD co-factor when inhibitors bind. Using the reversible inhibitor, D-amphetamine, as a model compound, a concentration-dependent change in the spectrum with clean isosbestic points was observed. The decrease in absorbance between 400 and 500 nm gave a dissociation constant for binding similar to the K(i) value. Anaerobic reduction yielded the semiquinone spectrum only and the midpoint potential was the same as the free enzyme. Full reduction was not possible with dithionite as the reductant, suggesting that the semiquinone-reduced couple had a much lower midpoint potential than the free enzyme. In contrast, with substrate, which reduces the enzyme on an equimolar basis, the semiquinone is never seen. In anaerobic stopped-flow experiments, amphetamine inhibits completely the reoxidation of the reduced enzyme in contrast to a substrate such as 2-phenylethylamine (the desmethyl analogue of amphetamine) that accelerates the rate 12-fold. The spectral changes in MAO-A permit the examination of inhibitor interaction with the redox co-factor. Stacking of the inhibitor and flavin rings constitutes part of the interaction but, taking into account other evidence, steric factors may be the clue to the differences between substrate and inhibitor.
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PMID:Interactions of D-amphetamine with the active site of monoamine oxidase-A. 1503 14

Structural studies on recombinant human monoamine oxidase A (hMAO-A) provides interesting insights on comparison with that determined for human MAO-B (hMAO-B) as well as comparison with that previously published for rat MAO-A. The active site cavity of hMAO-A is monopartite (as with rat MAO-A) while hMAO-B is a bipartite cavity. hMAO-A crystallizes as a monomeric form, in contrast to the dimeric forms exhibited by hMAO-B and rat MAO-A. All of the known MAO structures show nearly identical geometries around the covalent FAD sites. Differences in active site cavity structures occur away from the FAD site through conformational alterations (MAO-A's) and by changes in amino acid residues (hMAO-A and hMAO-B). Differences observed between human and rat MAO-A's raise questions regarding the appropriateness of the rat model in the development of MAO-A specific inhibitors as drugs for eventual human use.
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PMID:New insights into the structures and functions of human monoamine oxidases A and B. 1739 64

FAD (flavin adenine dinucleotide)-dependent monoamine oxidases play very important roles in many biological processes. A novel monoamine oxidase, named renalase, has been identified in human kidney recently and is found to be markedly reduced in patients with end-stage renal disease (ESRD). Here, we reported the identification of a renalase homologue from mouse, termed mMAO-C (mouse monoamine oxidase-C) after the monoamine oxidase-A and -B (MAO-A and -B). This gene locates on the mouse chromosome 19C1 and its coding region spans 7 exons. The deuced amino acid sequences were predicted to contain a typical secretive signal peptide and a conserved amine oxidase domain. Phylogenetic analysis and multiple sequences alignment indicated that mMAO-C-like sequences exist in all examined species and share significant similarities. This gene has been submitted to the NCBI GenBank database (Accession number: DQ788834). With expression vectors generated from the cloned mMAO-C gene, exogenous protein was effectively expressed in both prokaryotic and eukaryotic cells. Recombinant mMAO-C protein was secreted out of human cell lines, indicating the biological function of its signal peptide. Moreover, tissue expression pattern analysis revealed that mMAO-C gene is predominantly expressed in the mouse kidney and testicle, which implies that kidney and testicle are the main sources of renalase secretion. Shortly, this study provides an insight into understanding the physiological and biological functions of mMAO-C and its homologues in endocrine.
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PMID:Identification, expression and tissue distribution of a renalase homologue from mouse. 1784 19

The structure and mechanism of human monoamine oxidase B (MAO B) inhibition by hydrazines are investigated and compared with data on human monoamine oxidase A (MAO A). The inhibition properties of phenylethylhydrazine, benzylhydrazine, and phenylhydrazine are compared for both enzymes. Benzylhydrazine is bound more tightly to MAO B than to MAO A, and phenylhydrazine is bound weakly by either enzyme. Phenylethylhydrazine stoichiometrically reduces the covalent FAD moieties of MAO A and of MAO B. Molecular oxygen is required for the inhibition reactions, and the level of O2 consumption for phenylethylhydrazine is 6-7-fold higher with either MAO A or MAO B than for the corresponding reactions with benzylhydrazine or phenylhydrazine. Mass spectral analysis of either inhibited enzyme shows the major product is a single covalent addition of the hydrazine arylalkyl group, although lower levels of dialkylated species are detected. Absorption and mass spectral data of the inhibited enzymes show that the FAD is the major site of alkylation. The three-dimensional (2.3 A) structures of phenylethylhydrazine- and benzylhydrazine-inhibited MAO B show that alkylation occurs at the N(5) position on the re face of the covalent flavin with loss of the hydrazyl nitrogens. A mechanistic scheme is proposed to account for these data, which involves enzyme-catalyzed conversion of the hydrazine to the diazene. From literature data on the reactivities of diazenes, O2 then reacts with the bound diazene to form an alkyl radical, N2 and superoxide anion. The bound arylalkyl radical reacts with the N(5) of the flavin, while the dissociated diazene reacts nonspecifically with the enzyme through arylalkylradicals.
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PMID:Structural and mechanistic studies of arylalkylhydrazine inhibition of human monoamine oxidases A and B. 1842 26

New monoamine oxidase inhibitors were synthesized as indole analogues of a previously reported pyrrole series. Several compounds were potent MAO-A (12, 17, 19-22, 31, 36, and 37) or MAO-B (14, 20, 24, 38, 44, and 46) inhibitors, and had K(i) values in the nanomolar concentration range. In particular, 22 (K(i)=0.00092 microM, and SI=68,478) was exceptionally potent and selective as MAO-A inhibitor. In molecular modeling studies, compounds 22, 24, 44, and 46 positioned the indole ring into an aromatic cavity of MAO-A, and established pi-pi stacking interactions with Tyr407, Tyr444, and FAD cofactor. However, only compound 22 was able to form hydrogen bonds with FAD, a finding which was in accordance with its potent anti-MAO-A activity. Conversely, 22/MAOB complex was highly unstable during the MD simulation.
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PMID:Synthesis, structure-activity relationships and molecular modeling studies of new indole inhibitors of monoamine oxidases A and B. 1895 3


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