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)

Trichomonas vaginalis grown for 16 h in the presence of [(14)C]spermine formed a high intracellular pool of [(14)C]spermidine and a small but detectable pool of [(14)C]putrescine. When [(3)H]putrescine was added to the growth medium, a large intracellular pool of [(3)H]putrescine was found, but it was not further metabolized, confirming previous studies suggesting the absence of a forward-directed polyamine synthetic pathway in T. vaginalis. Spermidine:spermineN:(1)-acetyltransferase (SSAT) and polyamine oxidase enzyme activities were detected which collectively converted spermine to spermidine. Polyamine oxidase was localized in the hydrogenosome-enriched fraction, whereas SSAT was found predominantly in the cytosolic fraction. In the presence of saturating substrate, the trichomonad SSAT had an activity of 0. 39+/-0.09 nmol min(-1) (mg protein)(-1) (the mean of five analyses) and an apparent K:(m) for spermine of 1.7 microM. The enzyme was competitively inhibited by di(ethyl)norspermine with a K:(i) of 28 microM. Growth studies indicated that 50 microM di(ethyl)norspermine caused a 68% and 84% reduction in the intracellular concentrations of spermidine and spermine, respectively. The trichomonad polyamine oxidase required FAD as a cofactor and had an apparent K:(m) of 6.0 microM for N(1)-acetylspermine. The potential of bis(alkyl) polyamine analogues as antitrichomonad agents is discussed.
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PMID:Dependence of Trichomonas vaginalis upon polyamine backconversion. 1102 47

This paper reports the first purification method developed for the isolation of an homogeneous polyamine oxidase (PAO) from etiolated barley seedlings. The crude enzyme preparation was obtained after initial precipitation of the extract with protamine sulphate and ammonium sulphate. The enzyme was further purified to a final homogeneity (by the criteria of isoelectric focusing and SDS-PAGE) using techniques of low pressure chromatography followed by two FPLC steps. The purified yellow enzyme showed visible absorption maxima of a flavoprotein at 380 and 450 nm: the presence of FAD as the cofactor was further confirmed by measuring the fluorescence spectra. Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatography revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley PAO shows a high degree of similarity to that of maize PAO and to several other flavoprotein oxidases. The polyamines spermine and spermidine were the only two substrates of the enzyme with Km values 4 x 10(-5) and 3 x 10(-5) M and pH optima of 5.0 and 6.0, respectively. Barley polyamine oxidase is markedly inhibited by acridine dyes and hydrazines. Weak inhibition was observed with substrate analogues, aminoaldehydes, metal chelating agents and several other compounds.
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PMID:Barley polyamine oxidase: characterisation and analysis of the cofactor and the N-terminal amino acid sequence. 1170 21

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 (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.
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PMID:Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling. 1186 43

Polyamine oxidase is a FAD-dependent amine oxidase, which is constitutively expressed in nearly all tissues of the vertebrate organism. In 1985, N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was designed as a selective enzyme-activated irreversible inhibitor of polyamine oxidase (EC 1.5.3.11). It inactivates, at micromolar concentration and time-dependently, the enzyme in cells, as well as in all organs of experimental animals, without inhibiting other enzymes of polyamine metabolism. MDL 72527 served during nearly two decades as a unique tool in the elucidation of the physiological roles of polyamine oxidase. The compound has anticancer and contragestational effects, and it improves the anticancer effect of the ornithine decarboxylase inactivator (D,L)-2-(difluoromethyl)ornithine (DFMO). Profound depletion of the polyamine pools of tumour cells and effects on different components of the immune defence system are responsible for the anticancer effects of MDL 72527/DFMO combinations. Recently a direct cytotoxic effect of MDL 72527 at concentrations above those required for polyamine oxidase inactivation was observed. The induction of apoptosis by MDL 72527 was ascribed to its lysosomotropic properties. Therapeutic potentials of the apoptotic effect of MDL 72527 need to be explored. Polyamine oxidase is the last enzyme of the polyamine interconversion pathway that awaits the detailed elucidation of its structure and regulation. MDL 72527 should be useful as a lead in the development of inactivators which are selective for the isoforms of polyamine oxidase. Isozyme-selective inhibitors will give more profound insights into and reveal a diversity of specific functions of polyamine oxidase.
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PMID:The polyamine oxidase inactivator MDL 72527. 1245 62

In the polyamine back-conversion pathway, spermine and spermidine are first acetylated by spermidine/spermine N1 -acetyltransferase (SSAT) and then oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine respectively. Although PAO was first purified more than two decades ago, the protein has not yet been linked to genomic sequences. In the present study, we apply a BLAST search strategy to identify novel oxidase sequences located on human chromosome 10 and mouse chromosome 7. Homologous mammalian cDNAs derived from human brain and mouse mammary tumour were deduced to encode proteins of approx. 55 kDa having 82% sequence identity. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by approx. 30%, whereas spermidine increased 2-4-fold. Lysates of human PAO cDNA-transfected HEK-293 cells, but not vector-transfected cells, rapidly oxidized N1-acetylspermine to spermidine. Substrate specificity determinations with the lysate assay revealed a preference ranking of N1-acetylspermine= N1-acetylspermidine> N1,N12-diacetylspermine>>spermine; spermidine was not acted upon. This ranking is identical to that reported for purified PAO and distinctly different from the recently identified spermine oxidase (SMO), which prefers spermine over N1-acetylspermine. Monoethyl- and diethylspermine analogues also served as substrates for PAO, and were internally cleaved adjacent to a secondary amine. We deduce that the present oxidase sequences are those of the FAD-dependent PAO involved in the polyamine back-conversion pathway. In Northern blot analysis, PAO mRNA was much less abundant in HEK-293 cells than SMO or SSAT mRNA, and all three were differentially induced in a similar manner by selected polyamine analogues. The identification of PAO sequences, together with the recently identified SMO sequences, provides new opportunities for understanding the dynamics of polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically-relevant polyamine analogues and inhibitors.
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PMID:Genomic identification and biochemical characterization of the mammalian polyamine oxidase involved in polyamine back-conversion. 1247 80

N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was considered to be a selective inactivator of FAD-dependent tissue polyamine oxidase. Recently MDL 72527 was reported to induce apoptosis in transformed hematopoietic cells through lysosomotropic effects. Since it is the only useful inhibitor of polyamine oxidase available at present, the re-evaluation of its properties seemed important. Human colon carcinoma-derived SW480 cells and their lymph node metastatic derivatives (SW620) were chosen for our study because they differ in various aspects of polyamine metabolism but have similar polyamine oxidase activities. MDL 72527 inhibited cell growth in a concentration-dependent manner, depleted intracellular polyamine pools, and caused the accumulation of N1-acetyl derivatives of spermidine and spermine. SW620 cells were more sensitive to the drug than were SW480 cells. At 150 micromol/L MDL 72527, SW620 cells accumulated in S-phase of the cell cycle, showed decreased polyamine transport rate, and showed no increase of polyamine N1-acetyltransferase activity. In contrast, SW480 cells were not arrested in a particular phase of the cell cycle, showed enhanced polyamine uptake, and showed a mild induction of acetyltransferase. The results suggest that MDL 72527 retains its value as a selective tool in short-term experiments only at concentrations not exceeding those necessary for the inactivation of polyamine oxidase. At concentrations above 50 micromol/L and at exposure times longer than 24 h, it may derange cell functions nonspecifically, and thus blur the results of studies intended to elucidate polyamine oxidase functions.
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PMID:Cytotoxic effects of the polyamine oxidase inactivator MDL 72527 to two human colon carcinoma cell lines SW480 and SW620. 1248 49

The aminoacyl sequences of three regions of pure bovine N1-acetylated polyamine oxidase (PAO) were obtained and used to search GenBankTM. This led to the cloning and sequencing of a complete coding cDNA for murine PAO (mPAO) and the 5'-truncated coding region of the bovine pao (bpao) gene. A search of GenBankTM indicated that mpao maps to murine chromosome 7 as seven exons. The translated amino acid sequences of mpao and bpao have a -Pro-Arg-Leu peroxisomal targeting signal at the extreme C termini. A beta-alpha-beta FAD-binding motif is present in the N-terminal portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to corresponding sections of other flavoprotein amine oxidases, although the overall identity of aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of mpao was used as a probe to establish the relative transcription levels of this gene in various mature murine tissues and murine embryonic and breast tissues at different developmental stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a reasonable level. The mPAO so produced was purified to homogeneity and characterized. It was demonstrated definitively that PAO oxidizes N1-acetylspermine to spermidine and 3-acetamidopropanal and that it also oxidizes N1-acetylspermidine to putrescine and 3-acetamidopropanal. Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that oxidizes these N1-acetylated polyamines on the exo-side of their N4-amino groups. This enzyme is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of the N4-nitrogen. It differs also from mammalian spermine oxidase that oxidizes spermine (but not N1-acetylspermine or N1-acetylspermidine) at the exo-carbon of its N4-amino group. This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state kinetic properties of pure mPAO.
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PMID:Cloning, sequencing, and heterologous expression of the murine peroxisomal flavoprotein, N1-acetylated polyamine oxidase. 1266 Feb 32

In this report we show that recombinant Saccharomyces cerevisiae Fms1 protein is a polyamine oxidase that binds FAD with an FAD:Fms1 stoichiometry of 1:1. Biochemical characterization of Fms1 shows that it can oxidize spermine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine, but not spermidine. The products of spermine oxidation are spermidine and 3-aminopropanal. A kinetic analysis revealed that spermine, N(1)-acetylspermine, and N(1)-acetylspermidine are oxidized with similar efficiencies, while N(8)-acetylspermidine is a poor substrate. The data support a previous report, suggesting that Fms1 is responsible for the production of beta-alanine from spermine for the synthesis of pantothenic acid.
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PMID:Yeast Fms1 is a FAD-utilizing polyamine oxidase. 1267 Apr 77

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.
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PMID:Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase. 1503 14


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