Molecular cloning and nucleotide sequence of full‐length cDNA for ascorbate oxidase from cultured pumpkin cells

Abstract

A Agtl 1 cDNA expression library was constructed from size-fractionated poly(A)-rich RNA of cultured pumpkin cells. A full-length cDNA clone for ascorbate oxidase mRNA was selected from the library by screening with synthetic oligonucleotides designed from the amino-terminal sequence of ascorbate oxidase protein. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with that determined for the amino-terminal sequence of pumpkin ascorbate oxidase. The nucleotide sequence of the cDNA insert was found to contain an open reading frame of 579 codons corresponding to a signal peptide of 30 amino acids and the mature 549-residue ascorbate oxidase. Furthermore, it was found that the amino acid sequence deduced from the nucleotide sequence of the cDNA insert contained four potential Nglycosylation sites and copper-binding amino acid residues located in four regions where the sequence was identical or nearly identical to those of the other known blue multicopper oxidases Neurospora crassa laccase and human ceruloplasmin.Ascorbate oxidase is a copper-containing enzyme that occurs in many plant tissues such as pumpkin [l, 21, cucumber [3] and orange [4], and plays a role in secondary metabolism. The precise biological function of ascorbate oxidase has not been clarified, although it has been reported that the enzyme may participate in a redox system involving ascorbic acid IS]. However, the enzyme has recently been used as a valuable reagent for clinical and food analyses of L-ascorbic acid [6, 71.We have studied the ascorbate oxidase in cultured pumpkin cells and in a previous paper [8] reported that ascorbate oxidase activity rapidly increased during growth of pumpkin callus. We have found that ascorbate oxidase activity in cultured pumpkin cells can be markedly increased by adding copper, a prosthetic metal of the enzyme, and that this increase is accompanied by an increase in the enzyme protein, suggesting that copper controls the amount of ascorbate oxidase protein [9]. Furthermore, we have shown that ascorbate oxidase is secreted into the culture medium in the cell suspension cultures [lo] and that calcium ions markedly stimulate the secretion of ascorbate oxidase [ll]. Further studies are required to elucidate the molecular mechanisms responsible for regulating ascorbate oxidase gene expression.Recently, Ohkawa et al. [12] have isolated the cDNA for ascorbate oxidase from cucumber fruit tissues and determined the nucleotide sequence of the cDNA insert. So far, however, the cDNA for pumpkin ascorbate oxidase has not been cloned. In the present study, we constructed a cDNA expression library from poly(A)-rich RNA of cultured pumpkin cells and selected a full-length cDNA clone for ascorbateCorrespondence to

The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus

“…Saloheimo et al (1991) have shown that the molecular mass calculated from the ORF of the laccase gene from Phlebia radiata is 54-44 kDa and similar values were already established for Neurospora crassa (German et al, 1988), Coriolus hirsutus (Kojima et al, 1990) and Aspergillus nidulans (Aramoyo & Timberlake, 1990). The closely related blue copper oxidases (ascorbate oxidase) from plants also show a similar size of gene product (Esaka et al, 1990).…”

X‐ray crystallographic characterisation of type‐2‐depleted ascorbate oxidase from zucchini

“…The enzyme from Cucurbita pep0 medullosa (green zucchini) is a dimer of 140 kDa containing eight copper ions of three different spectroscopic forms classified as type-1, type-2 and type-3 [3]. Primary structures of ascorbate oxidase from cucumber [4] and pumpkin [5] have recently been reported.…”

Expression profiles of genes and enzymes related to ascorbic acid metabolism in fruits of Ziziphus jujuba Mill. ‘Jinsixiaozao’

“…One unit of enzyme was defined as the reduction of 1 mmol$L -1 cytochrome c per minute. The crude extraction and determination of ascorbate oxidase (AO) followed the method of Esaka [26] and dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), ascorbate peroxidase (APX) previously published methods [26,27] . Each treatment was replicated three times with each sample measured three times.…”