Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302.
Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.
Frébort
I
I
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Matsushita
K
K
Toyama
H
H
Lemr
K
K
Yamada
M
M
Adachi
O
O
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D004790:Enzyme Induction; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D013053:Spectrophotometry; D013379:Substrate Specificity
C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors