Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.
cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.
Ohkawa
J
J
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Okada
N
N
Shinmyo
A
A
Takano
M
M
D000595:Amino Acid Sequence; D001204:Ascorbate Oxidase / Q000235:genetics*; D001400:Azurin / Q000031:analogs & derivatives / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; C017079:mauC protein, Methylobacterium extorquens; D001400:Azurin; D004247:DNA; D010088:Oxidoreductases; D001204:Ascorbate Oxidase