- Characterization of a new enzyme oxidizing ω-amino group of aminocarboxyric acid, aminoalcohols and amines from Phialemonium sp. AIU 274
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A new enzyme exhibiting oxidase activity for ω-aminocarboxylic acids, ω-aminoalcohols, monoamines and diamines was found from a newly isolated fungal strain, Phialemonium sp. AIU 274. The enzyme also oxidized aromatic amines, but not l- and d-amino acids. The Vmax/Km value for hexylamine was higher than those for 6-aminoalcohol and 6-aminhexanoic acid in the aliphatic C6 substrates. In the aliphatic amines, the higher Vmax/Km values were obtained by the longer carbon chain amines. Thus, the enzyme catalyzed oxidative deamination of the ω-amino group in a wide variety of the ω-amino compounds and preferred medium- and long-chain substrates. The oxidase with such broad substrate specificity was first reported here. The enzyme contained copper, and the enzyme activity was strongly inhibited by isoniazid, iproniazid and semicarbazide, but not by clorgyline and pargyline. The enzyme was composed of two identical subunits of 75 kDa.
- Isobe, Kimiyasu,Sasaki, Tomoko,Aigami, Yuusuke,Yamada, Miwa,Kishino, Shigenobu,Ogawa, Jun
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- Mechanistic and structural analyses of the roles of active site residues in yeast polyamine oxidase Fms1: Characterization of the N195A and D94N enzymes
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Flavoprotein Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine in the biosynthetic pathway for pantothenic acid. The same reaction is catalyzed by the mammalian polyamine and spermine oxidases. The active site of Fms1 contains three amino acid residues positioned to interact with the polyamine substrate, His67, Asn195, and Asp94. These three residues form a hydrogen-bonding triad with Asn195 being the central residue. Previous studies of the effects of mutating His67 are consistent with that residue being important both for interacting with the substrate and for maintaining the hydrogen bonds in the triad [Adachi, M. S., Taylor, A. B., Hart, P. J., and Fitzpatrick, P. F. (2012) Biochemistry 51, 4888-4897]. The N195A and D94N enzymes have now been characterized to evaluate their roles in catalysis. Both mutations primarily affect the reductive half-reaction. With N 1-acetylspermine as the substrate, the rate constant for flavin reduction decreases ~450-fold for both mutations; the effects with spermine as the substrate are smaller, 20-40-fold. The kcat/Kamine- and kcat-pH profiles with N1-acetylspermine are only slightly changed from the profiles for the wild-type enzyme, consistent with the pKa values arising from the amine substrate or product and not from active site residues. The structure of the N195A enzyme was determined at a resolution of 2.0 ?. The structure shows a molecule of tetraethylene glycol in the active site and establishes that the mutation has no effect on the protein structure. Overall, the results are consistent with the role of Asn195 and Asp94 being to properly position the polyamine substrate for oxidation.
- Adachi, Mariya S.,Taylor, Alexander B.,Hart, P. John,Fitzpatrick, Paul F.
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p. 8690 - 8697
(2013/01/15)
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- Probing mammalian spermine oxidase enzyme-substrate complex through molecular modeling, site-directed mutagenesis and biochemical characterization
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Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.
- Tavladoraki, Paraskevi,Cervelli, Manuela,Antonangeli, Fabrizio,Minervini, Giovanni,Stano, Pasquale,Federico, Rodolfo,Mariottini, Paolo,Polticelli, Fabio
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experimental part
p. 1115 - 1126
(2012/01/19)
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- Mechanistic studies of human spermine oxidase: Kinetic mechanism and pH effects
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In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k3) of 49 s-1 and an apparent K d value of 48 μMat pH8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s-1, comparable to the kcat value of 6.6 s-1. The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order rate constant of 4.2 mM-1 s-1. At an aging time of 0.3 s, the reaction is biphasic with two second-order constants equal to 4.0 and 40 mM-1 s-1. Neither is equal to the kcat/KO2 value of 13 mM -1 s-1. These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The kcat/KM value for spermine exhibits a bell-shaped pH profile, with an average pKa value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k3 shows a pKa value of 7.4 for a group that must be unprotonated. The pKi-pH profiles for the competitive inhibitors N,N′-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pKa of ~7.4 in the active site are preferred for binding. 2009 American Chemical Society.
- Adachi, Mariya S.,Juarez, Paul R.,Fitzpatrick, Paul F.
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experimental part
p. 386 - 392
(2011/01/05)
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- Oxidation reaction of aliphatic amines and aminoalcohols in aqueous solution induced by argon arc plasma
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Argon arc plasma induced a powerful and stepwise oxidation reaction including conversion of methyl group to carboxyl group, oxidative cleavage of carbon-carbon bond, and oxidative deamination. Main active species were considered to be hydroxyl radicals by decomposition of water molecules.
- Takasaki, Michiaki,Harada, Kaoru
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p. 4463 - 4473
(2007/10/02)
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- FLAME-INDUCED OXIDATION OF ALIPHATIC AMINES IN AN AQUEOUS SOLUTION
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When a town gas-, a hydrogen-, and an ethylene-oxigen flame were blown against the surface of aqueous solution of aliphatic amines, the amines were effectively oxidized to afford amino acids.
- Nomoto, Shinya,Takasaki, Michiaki,Sakata, Naoko,Harada, Kaoru
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p. 3357 - 3360
(2007/10/02)
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- Oxidation of Polyamines by Fungal Enzymes
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Polyamine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Mucor, Penicillium, Rhizopus, Cylindrocarpon, Fusarium and Gibberella when they were grown in medium containing spermine or spermidine as the sole source of nitrogen.The maximal formation of the enzymes of Penicillium chrysogenum and Aspergillus terreus was observed in early stationary phase of growth, and thereafter, the enzymes disappeared with consumption of substrate.The oxidation products of spermine and spermidine by the two enzymes were identified as putrescine, 3-aminopropionaldehyde and H2O2.Therefore, the enzymes were characterized as a type of polyamine oxidase of rat liver.
- Yamada, Hideaki,Isobe, Kimiyasu,Tani, Yoshiki
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p. 2469 - 2476
(2007/10/02)
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