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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

1996; Elsevier BV; Volume: 1295; Issue: 1 Linguagem: Inglês

10.1016/0167-4838(96)00014-3

1878-1454

Ivo Frébort, Pavel Peč, Lenka Luhová, Hirohide Toyama, Kazunobu Matsushita, Shun Hirota, Teizo Kitagawa, Tamio Ueno, Yasuhisa Asano, Yasuo Kato, Osao Adachi,

Biochemical Acid Research Studies

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 × 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-nitrophenyl-hydrazone 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.