title: Two distinct quinoprotein amine oxidases are induced by n-butylamine in the mycelia of Aspergillus niger AKU 3302. Purification, characterization, cDNA cloning and sequencing.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
authors.forename: I
authors.initials: I
authors.lastname: Frébort
authors.forename: H
authors.initials: H
authors.lastname: Tamaki
authors.forename: H
authors.initials: H
authors.lastname: Ishida
authors.forename: P
authors.initials: P
authors.lastname: Pec
authors.forename: L
authors.initials: L
authors.lastname: Luhová
authors.forename: H
authors.initials: H
authors.lastname: Tsuno
authors.forename: M
authors.initials: M
authors.lastname: Halata
authors.forename: Y
authors.initials: Y
authors.lastname: Asano
authors.forename: Y
authors.initials: Y
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Matsushita
authors.forename: H
authors.initials: H
authors.lastname: Toyama
authors.forename: H
authors.initials: H
authors.lastname: Kumagai
authors.forename: O
authors.initials: O
authors.lastname: Adachi
mesh_terms: D006631:Amine Oxidase (Copper-Containing); D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002082:Butylamines / Q000494:pharmacology; D003300:Copper / Q000378:metabolism; D018076:DNA, Complementary; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011809:Quinones / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002082:Butylamines; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011809:Quinones; D012333:RNA, Messenger; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors; C032462:n-butylamine