Ohkawa J, Okada N, Shinmyo A, Takano M
Department of Fermentation Technology, Faculty of Engineering, Osaka University, Japan.
Proc Natl Acad Sci U S A. 1989 Feb;86(4):1239-43. doi: 10.1073/pnas.86.4.1239.
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.
从黄瓜（Cucumis sativus）果实mRNA构建的cDNA文库中分离出抗坏血酸氧化酶的cDNA克隆。用编码该酶NH2末端序列的合成寡核苷酸筛选该文库。对克隆的cDNA插入片段进行核苷酸序列分析，发现一个1761个碱基对的开放阅读框，编码一个由33个氨基酸组成的NH2末端信号肽和一个由554个氨基酸组成的成熟酶（Mr，62,258）。通过核苷酸序列分析推导的氨基酸序列与通过微量测序方法测定的纯化抗坏血酸氧化酶的NH2末端氨基酸序列一致。黄瓜抗坏血酸氧化酶含有四个富含组氨酸的区域，与其他多铜氧化酶（如粗糙脉孢菌漆酶和人铜蓝蛋白）的相应部分具有显著的序列同源性，并且在一定程度上与低分子量铜蛋白（如质体蓝素）具有序列同源性。此外，这些数据进一步支持了小蓝铜蛋白和多铜氧化酶是从同一祖先基因进化而来的假说。通过RNA印迹杂交分析，发现抗坏血酸氧化酶的mRNA在黄瓜果实组织中含量丰富，而在叶和根组织中表达水平很低。
