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 sou...

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Bibliographic Details
Published in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2000-04, Vol.64 (4), p.717-722
Main Authors: Koyanagi, T. (Osaka Univ., Suita (Japan). Inst. of Scientific and Industrial Research), Matsumura, K, Kuroda, S, Tanizawa, K
Format: Article
Language:English
Subjects:
ADN
AMINE OXIDASE
Amine Oxidase (Copper-Containing) - genetics
Amine Oxidase (Copper-Containing) - metabolism
AMINE OXYDASE
Amino Acid Sequence
AMINO OXIDASA
Base Sequence
Biological and medical sciences
Cations, Divalent
CLONACION MOLECULAR
CLONAGE MOLECULAIRE
Cloning, Molecular
COBRE
COPPER
copper amine oxidase
CUIVRE
DNA
DNA, Plant
Enzyme Activation
EXPRESION GENICA
EXPRESSION DES GENES
Fundamental and applied biological sciences. Psychology
GENE EXPRESSION
Genes, Plant
Genes. Genome
GENETIC ENGINEERING
GENIE GENETIQUE
INGENIERIA GENETICA
Molecular and cellular biology
MOLECULAR CLONING
Molecular genetics
Molecular Sequence Data
pea seedling
Pichia
Pichia pastoris
PISUM SATIVUM
Pisum sativum - enzymology
Pisum sativum - genetics
PLANTULAS
PLANTULE
QUINONAS
QUINONE
QUINONES
quinoprotein
SEEDLINGS
Seeds - enzymology
topa quinone
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Description
Summary: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 Pichla 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
ISSN:0916-8451
1347-6947
DOI:10.1271/bbb.64.717