Pea seedling amine oxidase

Turowski P. N. McGuirl M. A. Dooley D. M. Intramolecular electron transfer rate between active-site copper and topa quinone in pea seedling amine oxidase. J. Biol. Chem. 1993, 268, 17680-17682. 

With few exceptions [12,27], copper amine oxidases are homodimers in the native conformation. The molecular mass of the single subunit lies in the range 70-100 kDa. Most of the eukaryotic copper amine oxidases are glycoproteins. Their isoelectric point is usually slightly below pi 7.0, but some enzymes have pi > 7.0 such as pea seedling amine oxidase [12]. The amino acid composition is known for many amine oxidases and in the last decade, the complete amino acid sequence has been determined for a number of them. Some enzymes have been prepared in a crystalline form [12], four of them have been analyzed by X-ray diffraction with complete resolution of their three dimensional structure including detailed spatial conformation of the active site [28-31]. 

The copper can be reversibly removed from the active site by reaction with diethyldithiocarbamate under non-reducing conditions [86-88], or by cyanide after reduction by dithionate to Cu(I) [78]. The catalytic activity of the enzyme can be restored with high yield by addition of free Cu(II) ions to the apoenzyme [78,86]. In the case of pea seedling amine oxidase, addition of other bivalent metal ions does not lead to reactivation [86]. However the activity can be partially restored (from 15%) for the amine oxidase from bovine plasma by adding Co(II) [89]. Addition of Co(II) and Ni(II) can restore the original spectrum of the native enzyme with bovine serum amine oxidase reduced by dithionate [90]. 

Recent spectroscopic studies suggest, however, that this could happen only with the assistance of an active site lysyl residue [148]. Such a lysyl residue is found in the crystal structure of the active site of pea seedling amine oxidase [29], but is absent from E. coli amine oxidase [28]. The process of redox-active cofactor formation in phenethylamine oxidase and histamine oxidase of A. globiformis was recently analyzed by Raman spectroscopy using isotopic exchange. It was found that the oxygen on the... 

Quite recently, the cDNA coding for pea seedlings amine oxidase has been cloned and a heterologous expression system for the cloned enzyme... 

Wimmerova, M. and L. Macholan. 1999. Sensitive amperometric biosensor for the determination of biogenic and synthetic amines using pea seedlings amine oxidase A novel approach for enzyme immobilization. Biosensor. Bioelectron. 14 695-702. 

Tipping, A. J., and McPherson, M. J., 1995, Cloning and molecular analysis of the pea seedling copper amine oxidase, J. Biol. Chem. 270 16939916946. 

Vignevich, V., Dooley, D. M., Guss, J. M., Harvey, I., McGuirl, M. A., and Freeman, H. C., 1993, Crystallization and preliminary crystallographic characterization of copper-containing amine oxidase from pea seedlings, J. Mol. Biol. 229 2439245. 

Scheme 35 shows the multistep synthesis of optically pure tritium-labeled histamine, which involves an amine oxidase-catalyzed reaction as the first step [250], Diamine oxidase (DAMOX) from pea seedlings catalyze the oxidative deamination of diamines. DAMOX-catalyzed reactions of diamines in the synthesis of phenacyl azaheterocycles is shown in Scheme 36 [251-255],... 

The crystal structure of amine oxidase from Escherichia coli reveals that the C-terminal domain (440 amino acids) primarily has a /3-sandwich structure. This domain contains the active site with a type 2 copper center, the cofactor, and the dimerization contact site. Furthermore, there are three domains of approximately 100 amino acids length which have an a- and a /3-structure. The second and third of these smaller domains show marked sequence homology and are conserved in the amine oxidases of various organisms. The first of these smaller domains may, however, be lacking in some amine oxidases [28]. The structure of an eukariotic aminoxidase from pea seedling is seen in [121]. 

The crystal structure of CuAO has been solved from Escherichia coli (ECAO), pea seedling (PSAO), Arthrobacter globiformis (AGAO), Hansetmla polymorpha (HPAO), Pichia pastoris (PPLO), " bovine serum amine oxidase (BSAO), ° and human vascular adhesion protein (VAP-1). ... 

The results were further confirmed by resonance Raman spectrometry on comparing the spectra of phenylhydrazone and p-nitrophenylhydrazone of bovine plasma amine oxidase with the derivatized pentapeptides of the active site and the model compound. All these spectra showed great similarity in position (wavenumber) and spectral band intensity, while the spectrum of a PQQ model compound differed markedly [45]. Similar experiments confirmed the presence of topa quinone in porcine kidney, pea seedling and Arthrobacter PI amine oxidases. Moreover, the experimental data obtained for intact enzymes excluded the possibility of an artificial topa quinone formation during the proteolysis and peptide isolation [45]. 

Later, the presence of topa quinone was accordingly confirmed in the amine oxidases from porcine serum and kidney and pea seedling by resonance Raman spectrometry of active-site labeled peptides [48]. Comparison of amino acid sequences of these peptides with the sequences of those from bovine plasma and H. polymorpha amine oxidases demonstrated the presence of a consensus sequence Asp-TPQ-Asp/Glu as shown in Fig. (1). Using the pH-dependent shift of the absorption maximum of the enzyme p-nitrophenylhydrazone, which is considered to be a reliable indirect proof, the presence of topa quinone was also shown... 

Recently, the cofactor peptides have also been isolated from semicarbazide-sensitive amine oxidases purified from bovine and porcine aortas [52], sequenced and confirmed to contain the topa quinone. The same topa quinone consensus sequence was also found in the primary structures of amine oxidases from human kidney [53], human retina [54] and rat colon [55], so called amiloride-binding proteins , and amine oxidase from human placenta [56] that shows 81% identity with bovine plasma amine oxidase [57], bovine lung amine oxidase [58], and amine oxidases from pea and lentil seedlings [59,60], chick pea seedlings [61], and Arabidopsis thaliana [62] obtained by the molecular cloning of respective DNAs. 

Fig. (2). Ribbon diagram of the three-dimensional crystal structure of copper amine oxidase from Escherichia coli [28], Similar structures of amine oxidases from pea seedlings [29], Arthrobacter globiformis [30] and Hansenula pclymorpha [31 ] lack the domain D1.

The above reaction mechanism has been confirmed by experimental studies. The stoichiometric formation of the aldehyde, H2O2, and ammonia from several substrates has been demonstrated with amine oxidases purified from beef plasma (ISl) and pea seedlings 1117,1S8), and with diamine oxidase purihed from hog kidney acetone powder 1 4)-... 

An amine oxidase has been purified from pea seedlings by Mann and associates (197,198). The purified enzyme catalyzed the oxidation not only of diamines but also of phenylalkylamines, aliphatic monoamines, and of L-lysine. The rates of oxidation for the latter three were less than for the diamines. The oxidation of all compounds was similarly affected by cyanide and semicarbazide. The conclusion reached was that a single enzyme is involved which is less specific than animal diamine oxidase. 

Suspensions of Achromobacter have been shown to oxidize putrescine, histamine, and isoam amine (17S). The oxidation is blocked by semicar-bazide (111). Separate enzymes w e suggested from these studies although it was recognized that their behaviors were similar to those of mammalian diamine oxidase. These results, as well as those with the purified pea seedling enzyme discussed above, arecited by Foutset of. (119) as indicative that the ability of mammalian diamine oxidase to react with monoamines also holds for diamine oxidase from other sources. 

The amine oxidase purified from pea seedlings 128) was inhibited by sodium diethyldithiocarbamate, potassium ethylxanthate, and salicylal-doxime, all of which are usually considered as inhibitors of copper-containing enzymes. The preparation of the enz5une contained both copper and manganese (0.03-0.06% based on protein content). It is suggested that the plant enzyme may be a metalloprotein. 

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