Cloning and sequencing of phenylethylamine oxidase from Arthrobacter globiformis and implication of Tyr-382 as the precursor to its covalently bound quinone cofactor.
The gene of Arthrobacter globiformis encoding a quinoprotein, phenylethylamine oxidase, has been cloned and sequenced. In the deduced amino acid sequence comprising 638 residues is a tetrapeptide sequence, Asn-Tyr-Asp-Tyr, which has been found to be highly conserved in other copper amine oxidase. Mutation of the former Tyr (Tyr-382) of the recombinant enzyme into Phe resulted in the complete loss of catalytic activity and disappearance of the quinone compound that is specifically detected in the wild-type enzyme, suggesting that Tyr-382 is the precursor to the covalently-bound cofactor, most probably topa quinone. Furthermore, the expression of the active, quinone-containing enzyme in Escherichia coli cells was markedly dependent on the presence of Cu2+ ions in the culture medium, and the inactive, Cu2(+)-deficient enzyme produced without Cu2+ ions could be converted to the active quinone form by reconstitution with Cu2+ ions.
Tanizawa
K
K
Institute of Scientific and Industrial Research, Osaka University, Japan.
Matsuzaki
R
R
Shimizu
E
E
Yorifuji
T
T
Fukui
T
T
D006631:Amine Oxidase (Copper-Containing) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine
D017931:DNA Primers; D011809:Quinones; D011994:Recombinant Proteins; D014443:Tyrosine; C086534:phenylethylamine oxidase; D006631:Amine Oxidase (Copper-Containing)