2 DISTINCT QUINOPROTEIN AMINE OXIDASES ARE INDUCED BY N-BUTYLAMINE INTHE MYCELIA OF ASPERGILLUS-NIGER AKU-3302 PURIFICATION, CHARACTERIZATION, CDNA CLONING AND SEQUENCING

Two distinct quinoprotein amine oxidases were found in Aspergillus nig er mycelia grown on n-butylamine medium and purified using chromatogra phic techniques. The respective enzymes were termed AO-I, which had al ready been isolated, and AO-II, a new enzyme found in this study. HPLC indicated that their molecular masses are 150 kDa and 80 kDa, respect ively. On SDS/PAGE, the enzymes gave a similar but distinct mobility, which corresponds to 75 kDa for the subunit of dimeric AO-I and 80 kDa for monomeric AO-II. The absorption spectra of both enzymes were diff erent from each other; the absorption maxima in the visible region wer e at 490 nm for AO-I and 420 nm for AO-II. The enzymes showed positive quinone staining, comparable substrate specificity, and sensitivity t o 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 pattern s, with the exception of two additional bands for AO-II. Among the pep tides obtained from digestion of AO-II, peptides with sequences corres ponding to the N-terminal part of AO-I were detected. Polyclonal antib odies raised against AO-I and AO-II recognized both enzymes, but with different specificities. Using precipitation with AO-I, the antibody p repared against AO-II was purified and was shown to be specific only f or AO-II. The cDNA of AO-I was cloned and sequenced. A highly conserve d tetrapeptide sequence, Asn-Tyr-Glu-Tyr, was identified in which the first tyrosine residue (Tyr404) that could be converted to topa quinon e was present in the 670-residue deduced amino acid sequence. Northern blot analysis indicated that AO-I was highly expressed in A. niger gr own 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.