Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases.

Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.

Cytokinins as inhibitors of plant amine oxidase.

The interaction of pea seedling amine oxidase with cytokinins was examined to probe a possible connection between cytokinin oxidase and amine oxidase by determining whether cytokinins are substrates or inhibitors of the latter. Kinetic measurements suggest that cytokinins are weak competitive inhibitors of amine oxidase while their behaviour as substrates was not observed. The absence of enzymatic activity with cytokinins as substrates denies the identity or even any similarity of these two enzymes which was previously considered [Hare, P.D. and van Staden, J. (1994) J. Physiol. Plant., 91, 128]. From the values of the inhibition constants obtained it seems unlikely that cytokinins take part in the regulation of amine oxidase activity in vivo. Their inhibitory effect on amine oxidase may be similar to that of some alkaloids studied earlier.

Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes.

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Two distinct quinoprotein amine oxidases are induced by n-butylamine in the mycelia of Aspergillus niger AKU 3302. Purification, characterization, cDNA cloning and sequencing.

Two distinct quinoprotein amine oxidases were found in Aspergillus niger mycelia grown on n-butylamine medium and purified using chromatographic techniques. The respective enzymes were termed AO-I, which had already been isolated, and AO-II, a new enzyme found in this study. HPLC indicated that their molecular masses are 150 kDa and 80 kDa, respectively. On SDS/PAGE, the enzymes gave a similar but distinct mobility, which corresponds to 75 kDa for the subunit dimeric AO-I and 80 kDa for monomeric AO-II. The absorption spectra of both enzymes were different from each other; the absorption maxima in the visible region were at 490 nm for AO-I and 420 nm for AO-II. The enzymes showed positive quinone staining, comparable substrate specificity, and sensitivity to inhibitors typical for copper/topa quinone-containing amine oxidases, but they had different copper contents and also differed in their N-terminal sequences. Their peptide maps showed almost identical patterns, with the exception of two additional bands for AO-II. Among the peptides obtained from digestion of AO-II, peptides with sequences corresponding to the N-terminal part of AO-I were detected. Polyclonal antibodies raised against AO-I and AO-II recognized both enzymes, but with different specificities. Using precipitation with AO-I, the antibody prepared against AO-II was purified and was shown to be specific only for AO-II. The cDNA of AO-I was cloned and sequenced. A highly conserved tetrapeptide sequence, Asn-Tyr-Glu-Tyr, was identified in which the first tyrosine residue (Tyr404) that could be converted to topa quinone was present in the 670-residue deduced amino acid sequence. Northern blot analysis indicated that AO-I was highly expressed in A. niger grown on n-butylamine as a single nitrogen source. Genomic Southern blot analysis confirmed that both enzymes are likely to be encoded by the same gene.

Comparison of kinetic properties between plant and fungal amine oxidases.

Kinetic properties of novel amine oxidases isolated from a mold Aspergillus niger AKU 3302 were compared to those of typical plant amine oxidase from pea seedling (EC 1.4.3.6). Pea amine oxidase showed highest affinity with diamines, such as putrescine and cadaverine, while fungal enzymes oxidized preferably n-hexylamine and tyramine. All enzymes were inhibited by carbonyl reagents, copper chelating agents, some substrate analogs and alkaloids, but there were quite significant differences in the sensitivity and inhibition modes. Aminoguanidine, which strongly inhibited pea amine oxidases showed only little effect on fungal enzymes. Substrate analogs such as 1.5-diamino-3-pentanone and 1-amino-3-phenyl-3-propanone, which were potent competitive inhibitors of pea amine oxidases, inhibited fungal enzymes much more weakly and non competitively. Also various alkaloids behaving as competitive inhibitors of pea amine oxidase inhibited the fungal enzymes non competitively. Very surprising was the potent inhibition of fungal enzymes by artificial substrates of pea amine oxidases, E- and Z-1,4-diamino-2-butene. The relationships between the different inhibition modes and possible binding at the active site are discussed.

Inhibition of copper/quinoprotein amine oxidases from Aspergillus niger by benzophenanthridine alkaloids.

Inhibition of copper/quinoprotein amine oxidases (EC 1.4.3.6), AO-I (dimer 2 x 75 kDa) and AO-II (monomer 80 kDa), from the fungus Aspergillus niger by benzophenanthridine alkaloids sanguinarine, chelerythrine, and fagaronine were studied. For both amine oxidases the alkaloids showed reversible noncompetitive inhibition of n-hexylamine oxidation with Ki 0.6, 0.9 and 2.8 mM for sanguinarine, chelerythrine, and fagaronine, respectively. The values of the inhibition constants corresponded to pKR+ values for the iminium ion/pseudobase equilibrium of the alkaloids. Since thio-compounds protected the enzymes against this inhibition, the inhibition effect was ascribed to the interaction with a sulfhydryl group essential for the enzymatic activity.

Active-site covalent modifications of quinoprotein amine oxidases from Aspergillus niger. Evidence for binding of the mechanism-based inhibitor, 1,4-diamino-2-butyne, to residue Lys356 involved in the catalytic cycle.

Interactions of two distinct quinoprotein amine oxidases from Aspergillus niger, AO-I and AO-II, with active-site covalent modifiers have been investigated. Both enzymes are inhibited similarly by phenylhydrazine or p-nitrophenylhydrazine, forming an orange Schiff base with a carbonyl group of topaquinone cofactor. Modification of histidyl and tyrosyl residues by diethylpyrocarbonate and sulfhydryl groups by 5,5'-dithio-bis-(2-nitrobenzoic acid) and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole have been described. A substrate analog, 1,4-diamino-2-butyne, was found to function as a mechanism-based inhibitor. It shows both substrate saturation kinetics and time-dependent irreversible inhibition caused by formation of pyrrole bound to the active site. The pyrrole formation was confirmed spectrophotometrically by reaction with Ehrlich's reagent at 525 nm. Inhibition by 1,4-diamino-2-butyne produces a new maximum in the absorption spectra of AO-I and AO-II at 310 nm and 306 nm, respectively. Inactivated AO-I was digested by proteases; labeled peptides were purified by C18 HPLC and sequenced by Edman degradation. Data reveal the evidence that 1,4-diamino-2-butyne reacts with the epsilon-amino group of the Lys356 residue in the sequence Lys-Met-Pro-Asn-Ala of Aspergillus niger amine oxidase AO-I.