Vitu Elvira, Bentzur Moran, Lisowsky Thomas, Kaiser Chris A, Fass Deborah
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
J Mol Biol. 2006 Sep 8;362(1):89-101. doi: 10.1016/j.jmb.2006.06.070. Epub 2006 Aug 8.
The ERV/ALR sulfhydryl oxidase domain is a versatile module adapted for catalysis of disulfide bond formation in various organelles and biological settings. Its four-helix bundle structure juxtaposes a Cys-X-X-Cys dithiol/disulfide motif with a bound flavin adenine dinucleotide (FAD) cofactor, enabling transfer of electrons from thiol substrates to non-thiol electron acceptors. ERV/ALR family members contain an additional di-cysteine motif outside the four-helix-bundle core. Although the location and context of this "shuttle" disulfide differs among family members, it is proposed to perform the same basic function of mediating electron transfer from substrate to the enzyme active site. We have determined by X-ray crystallography the structure of AtErv1, an ERV/ALR enzyme that contains a Cys-X4-Cys shuttle disulfide and oxidizes thioredoxin in vitro, and compared it to ScErv2, which has a Cys-X-Cys shuttle and does not oxidize thioredoxin at an appreciable rate. The AtErv1 shuttle disulfide is in a region of the structure that is disordered and thus apparently mobile and exposed. This feature may facilitate access of protein substrates to the shuttle disulfide. To test whether the shuttle disulfide region is modular and can confer on other enzymes oxidase activity toward new substrates, we generated chimeric enzyme variants combining shuttle disulfide and core elements from AtErv1 and ScErv2 and monitored oxidation of thioredoxin by the chimeras. We found that the AtErv1 shuttle disulfide region could indeed confer thioredoxin oxidase activity on the ScErv2 core. Remarkably, various chimeras containing the ScErv2 Cys-X-Cys shuttle disulfide were found to function efficiently as well. Since neither the ScErv2 core nor the Cys-X-Cys motif is therefore incapable of participating in oxidation of thioredoxin, we conclude that wild-type ScErv2 has evolved to repress activity on substrates of this type, perhaps in favor of a different, as yet unknown, substrate.
ERV/ALR巯基氧化酶结构域是一种多功能模块，适用于催化各种细胞器和生物环境中的二硫键形成。其四螺旋束结构将一个Cys-X-X-Cys二硫醇/二硫化物基序与一个结合的黄素腺嘌呤二核苷酸（FAD）辅因子并列，从而使电子从硫醇底物转移到非硫醇电子受体。ERV/ALR家族成员在四螺旋束核心之外还含有一个额外的二半胱氨酸基序。尽管这种“穿梭”二硫键在家族成员中的位置和背景有所不同，但据推测它执行相同的基本功能，即介导电子从底物转移到酶活性位点。我们通过X射线晶体学确定了AtErv1的结构，AtErv1是一种ERV/ALR酶，含有一个Cys-X4-Cys穿梭二硫键，并且在体外氧化硫氧还蛋白，同时将其与ScErv2进行比较，ScErv2具有一个Cys-X-Cys穿梭结构，且不以可观的速率氧化硫氧还蛋白。AtErv1穿梭二硫键位于结构中无序的区域，因此显然是可移动且暴露的。这一特征可能有助于蛋白质底物接近穿梭二硫键。为了测试穿梭二硫键区域是否具有模块化，以及是否能够赋予其他酶对新底物的氧化酶活性，我们构建了嵌合酶变体，将AtErv1和ScErv2的穿梭二硫键和核心元件结合起来，并监测嵌合体对硫氧还蛋白的氧化作用。我们发现AtErv1穿梭二硫键区域确实能够赋予ScErv2核心硫氧还蛋白氧化酶活性。值得注意的是，还发现各种含有ScErv2 Cys-X-Cys穿梭二硫键的嵌合体也能高效发挥作用。由于ScErv2核心和Cys-X-Cys基序都并非不能参与硫氧还蛋白的氧化，我们得出结论，野生型ScErv2已经进化到抑制对这类底物的活性，也许是为了有利于另一种尚未知晓的不同底物。
