Portnoy M E, Rosenzweig A C, Rae T, Huffman D L, O'Halloran T V, Culotta V C
Departments of Environmental Health Sciences and Biochemistry, Johns Hopkins University School of Public Health, Baltimore, Maryland 21205, USA.
J Biol Chem. 1999 May 21;274(21):15041-5. doi: 10.1074/jbc.274.21.15041.
Saccharomyces cerevisiae Atx1p represents a member of the family of metallochaperone molecules that escort copper to distinct intracellular targets. Atx1p specifically delivers copper to the Ccc2p copper transporter in the Golgi. Additionally, when overproduced, Atx1p substitutes for superoxide dismutase 1 in preventing oxidative damage; however the mechanistic overlap between these functions is unresolved. The crystal structure of Atx1p has been solved recently. By examining a surface electrostatic potential distribution, multiple conserved lysines are revealed on one face of Atx1p. An additional conserved lysine (Lys65) lies in close proximity to the metal binding site. Through site-directed mutagenesis, residues in the metal binding region including Lys65 were found to be necessary for both copper delivery to Ccc2p and for Atx1p antioxidant activity. Copper trafficking to Ccc2p also relied on the lysine-rich face of Atx1p. Surprisingly however, elimination of these lysines did not inhibit the antioxidant activity of Atx1p. We provide evidence that Atx1p does not suppress oxidative damage by a metallochaperone mechanism but may directly consume superoxide. Purified Cu-Atx1p reacts noncatalytically with superoxide anion in vitro. We conclude that the copper-trafficking and antioxidant functions of Atx1p arise from chemically and structurally distinct attributes of this metallochaperone.
酿酒酵母Atx1p是金属伴侣分子家族的成员之一，该家族负责将铜运送到不同的细胞内靶点。Atx1p特异性地将铜输送到高尔基体中的Ccc2p铜转运蛋白。此外，当过量表达时，Atx1p在预防氧化损伤方面可替代超氧化物歧化酶1；然而，这些功能之间的机制重叠尚未解决。Atx1p的晶体结构最近已得到解析。通过检查表面静电势分布，在Atx1p的一侧发现了多个保守的赖氨酸。另一个保守的赖氨酸（Lys65）位于靠近金属结合位点的位置。通过定点诱变，发现金属结合区域中的残基（包括Lys65）对于将铜输送到Ccc2p以及Atx1p的抗氧化活性都是必需的。铜向Ccc2p的转运也依赖于Atx1p富含赖氨酸的一侧。然而，令人惊讶的是，去除这些赖氨酸并没有抑制Atx1p的抗氧化活性。我们提供的证据表明，Atx1p不是通过金属伴侣机制抑制氧化损伤，而是可能直接消耗超氧化物。纯化的Cu-Atx1p在体外与超氧阴离子发生非催化反应。我们得出结论，Atx1p的铜转运和抗氧化功能源于这种金属伴侣在化学和结构上的不同特性。
