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.
Frébort
I
I
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Pec
P
P
Luhová
L
L
Toyama
H
H
Matsushita
K
K
Hirota
S
S
Kitagawa
T
T
Ueno
T
T
Asano
Y
Y
Kato
Y
Y
Adachi
O
O
D006631:Amine Oxidase (Copper-Containing); D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D003067:Coenzymes / Q000032:analysis / Q000737:chemistry*; D016384:Consensus Sequence; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D010659:Phenylhydrazines / Q000737:chemistry; D017421:Sequence Analysis
D003067:Coenzymes; D010446:Peptide Fragments; D010659:Phenylhydrazines; C005307:4-nitrophenylhydrazine; D018698:Glutamic Acid; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors