From the Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
J Biol Chem. 2014 Mar 28;289(13):9396-407. doi: 10.1074/jbc.M113.534446. Epub 2014 Feb 13.
Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.
维生素 K 环氧化物还原酶复合物亚基 1（VKORC1）在维生素 K 循环中还原维生素 K 环氧化物，用于参与各种生物功能的蛋白质的翻译后修饰。然而，与 VKORC1 具有约 50%蛋白质同源性的平行酶 VKORC1 样 1（VKORC1L1）的生理功能尚不清楚。在这里，我们使用荧光蛋白酶保护（FPP）测定法和体内基于细胞的活性测定法来确定这两种酶的结构和功能差异。我们表明，体内 VKORC1L1 还原维生素 K 环氧化物以有效地支持维生素 K 依赖性羧化作用，就像 VKORC1 一样。然而，FPP 测定法表明，与 VKORC1 不同，VKORC1L1 是一种具有四个跨膜结构域的蛋白质，其两端都位于细胞质中。此外，不需要 VKORC1 活性的保守环半胱氨酸对于 VKORC1L1 的活性位点再生是必不可少的。在 VKORC1L1 和 VKORC1 之间进行结构域交换的结果表明，正是 VKORC1L1 的整体结构独特地允许通过保守环半胱氨酸进行活性位点再生。中间二硫化物捕获结果证实了 VKORC1L1 的活性位点还原的分子内电子转移途径。我们的结果使我们能够提出 VKORC1L1 的四个保守半胱氨酸的协同作用用于活性位点再生；第二个环半胱氨酸，半胱氨酸-58，攻击活性位点二硫化物，与半胱氨酸-139 形成中间二硫化物；第一个环半胱氨酸，半胱氨酸-50，攻击中间二硫化物导致活性位点还原。VKORC1L1 和 VKORC1 之间的不同膜拓扑结构和反应机制表明这两种蛋白质可能具有不同的生理功能。
