title: Gene organization and molecular modeling of copper amine oxidase from Aspergillus niger: re-evaluation of the cofactor structure.
abstract: Amine oxidase AO-I from Aspergillus niger AKU 3302 has been reported to contain topa quinone (TPQ) as a cofactor; however, analysis of the p-nitrophenylhydrazine-derivatized enzyme and purified active site peptides showed the presence of a carboxylate ester linkage of TPQ to a glutamate. The catalytic functionality of such a cross-linked cofactor has recently been shown unlikely by spectroscopic and voltammetric studies on synthesized model compounds. We have obtained resonance Raman spectra of native and substrate-reduced AO-I demonstrating that the catalytically active cofactor is unmodified TPQ. The primary structure of the enzyme (GenBank acc. no. U31869) has been reviewed and updated by repeated isolation and sequencing of AO-I cDNA. This allowed rectification of several errors that account for previously reported low homology to other amine oxidases in the regions around copper binding histididyl residues. The results were confirmed by cloning the ao-1 structural gene (GenBank acc. no. AF362473). Analysis of the gene 5'-upstream region of the gene revealed potential binding sites for an analog of NIT2, the nitrogen metabolism regulatory protein found in Neurospora crassa and other fungi. The molecular structure of AO-I was modeled by a comparative method using published crystal structures of amine oxidases as templates.
authors.affiliation: Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, CZ-783 71 Olomouc, Czech Republic. frebort@risc.upol.cz
authors.forename: Ivo
authors.initials: I
authors.lastname: Frébort
authors.forename: Marek
authors.initials: M
authors.lastname: Sebela
authors.forename: Shun
authors.initials: S
authors.lastname: Hirota
authors.forename: Mamoru
authors.initials: M
authors.lastname: Yamada
authors.forename: Hisanori
authors.initials: H
authors.lastname: Tamaki
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Kumagai
authors.forename: Osao
authors.initials: O
authors.lastname: Adachi
authors.forename: Pavel
authors.initials: P
authors.lastname: Pec
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D005796:Genes; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology; D010659:Phenylhydrazines / Q000737:chemistry; D016415:Sequence Alignment; D013059:Spectrum Analysis, Raman
chemical_list: D003067:Coenzymes; D018076:DNA, Complementary; D010659:Phenylhydrazines; D018698:Glutamic Acid; D006639:Histidine; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D006631:Amine Oxidase (Copper-Containing)