Enzymological and structural biology investigation on the fuction of the vitamin K cycle

在DFG支持的研究项目中，我们于2004年确定维生素K环氧化物还原酶复合物亚基1（VKORC 1）为维生素K循环的中心组分（Rost 2004）。自2012年以来，再次得到DFG的支持，我们能够定位VKORC 1分子的天然华法林结合位点，表征华法林耐药患者中该结合位点内的结构变化，并确定VKCFD 2患者中所有维生素K依赖性凝血因子缺乏的分子原因。此外，我们深入研究了人VKORC 1的酶学性质。因此，从根本上改进了源于20世纪60年代的体外活性测定。我们现在申请的研究项目将集中于进一步表征VKORC 1和VKORC 1 L1，特别是维生素K和华法林的结合及其抑制机制。此外，我们还想研究维生素K循环的第二种必需酶，γ-谷氨酰羧化酶（GGCX）。这种酶负责所有维生素K依赖性蛋白的翻译后修饰。这种酶的突变同样导致VKCFD。通过最近建立的HEK 293 GGCX敲除细胞系，我们希望研究GGCX的功能和生物学相关性。总之，这些研究将大大有助于了解维生素K循环及其在不同器官系统中的作用。

In context of a DFG-supported research project, in 2004 we identified vitamin K epoxide reductase complex subunit 1 (VKORC1) as the central component of the vitamin K cycle (Rost 2004). Since 2012, again supported by the DFG, we were able to localize the native warfarin binding site of the VKORC1 molecule, characterize structural changes within this binding site in patients with warfarin resistance, and identify the molecular cause of deficiency of all vitamin K dependent coagulation factors in patients suffering from VKCFD2. Furthermore, we in depth characterized the enzymological properties human VKORC1. For this, the in vitro activity assay originating from the 1960s was fundamentally improved.The research project, on which we apply now, will concentrate on further characterization of VKORC1 and VKORC1L1, particularly on binding of vitamin K and warfarin and the mechanism of inhibition. Furthermore, we want to investigate the second essential enzyme of the vitamin K cycle, gamma-glutamyl-carboxylase (GGCX). This enzyme is responsible for posttranslational modification of all vitamin K dependent proteins. Mutations in this enzyme likewise lead to VKCFD. By means of a recently established HEK293 GGCX knock-out cell line we want to study function and biological relevance of GGCX. Taken together, these studies will contribute significantly to the knowledge on the vitamin K cycle and its role in different organ systems.