COPPER AMINE OXIDASE - HETEROLOGOUS EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF AN ACTIVE ENZYME IN SACCHAROMYCES-CEREVISIAE
COPPER AMINE OXIDASE - HETEROLOGOUS EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF AN ACTIVE ENZYME IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1021/bi00190a019
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发表时间：
1994-06-21
期刊：
影响因子：
2.9
通讯作者：
KLINMAN, JP
中科院分区：
文献类型：
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作者：
CAI, DY;KLINMAN, JP
来自甲基营养酵母多形汉逊酵母的铜胺氧化酶基因已在 ADHI 启动子的控制下在酿酒酵母中表达，并且重组蛋白纯化至接近均质。重组酶在催化甲胺氧化方面与天然酶一样活跃。我们通过氧化还原循环染色和用羰基试剂滴定证明它是一种醌蛋白。重组胺氧化酶及其苯肼衍生物的吸收光谱特性与其他铜胺氧化酶非常相似。该酶的辅因子是 2,4,5-三羟基苯丙氨酸 (topa) 醌，对硝基苯腙加合物的 lambda(max) 的 pH 依赖性变化证明了这一点。活性位点肽和 DNA 衍生蛋白质序列的比对揭示了酪氨酸残基是托帕醌的前体，这与其他铜胺氧化酶的发现一致。本文提供的所有证据表明，异源表达的铜胺氧化酶蛋白在酿酒酵母中进行翻译后加工，形成具有完整辅因子的活性酶。尽管酿酒酵母无法利用胺作为氮源，但这种情况还是发生了。讨论了本研究对托帕醌生物发生机制的影响。
A copper amine oxidase gene from a methylotrophic yeast Hansenula polymorpha has been expressed in Saccharomyces cerevisiae under the control of the ADHI promoter and the recombinant protein purified to near homogeneity. The recombinant enzyme is as active as the native enzyme in catalyzing methylamine oxidation. We demonstrate that it is a quinoprotein by redox-cycling staining and titrations with carbonyl reagents. The absorption spectral properties of the recombinant amine oxidase and its phenylhydrazine derivative are very similar to those of other copper amine oxidases. The cofactor in the enzyme is 2,4,5-trihydroxyphenylalanine (topa) quinone, as demonstrated by the pH-dependent shift in the lambda(max) of the p-nitrophenylhydrazone adduct. Alignment of an active-site peptide and DNA-derived protein sequences reveals a tyrosine residue as the precursor to topa quinone, consistent with findings with other copper amine oxidases. All evidence presented herein indicates that the heterologously expressed copper amine oxidase protein is processed posttranslationally in S. cerevisiae to form an active enzyme with an intact cofactor. This occurs despite an inability of S. cerevisiae to utilize amines as a nitrogen source. The implications of this study for the mechanism of topa quinone biogenesis are discussed.