Molecular cloning and heterologous expression of pea seedling copper amine oxidase.
The cDNA coding for copper amine oxidase has been cloned from etiolated pea seedlings (Pisum sativum). The deduced amino acid sequence, consisting of 674 residues including the signal peptide, agreed well with those reported for the enzymes from a different cultivar of P. sativum and other plant sources, except for several evolutionary replacements located mostly on the molecular surface. A heterologous expression system for the cloned pea enzyme was constructed with the yeast Pichia pastoris, using the AOX1 promoter and the yeast alpha-factor secretion signal. Adding copper to the culture medium increased the secretion of an active, quinone-containing enzyme. Furthermore, the inactive enzyme produced in a copper-deficient medium was activated considerably by subsequent incubation with excess cupric ions. These results strongly suggest that the Tyr-derived redox cofactor, 2,4,5-trihydroxyphenylalanylquinone (topa quinone, TPQ), is produced in the plant enzyme by post-translational modification that proceeds through the copper-dependent, self-processing mechanism, as in the enzymes from bacteria and yeast.
Koyanagi
T
T
Department of Structural Molecular Biology, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
Matsumura
K
K
Kuroda
S
S
Tanizawa
K
K
D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D003300:Copper; D018744:DNA, Plant; D004789:Enzyme Activation; D015870:Gene Expression; D017343:Genes, Plant; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics; D010843:Pichia; D012639:Seeds / Q000201:enzymology
D002413:Cations, Divalent; D018744:DNA, Plant; D003300:Copper; D006631:Amine Oxidase (Copper-Containing)