The Molybdenum Cofactor: Biosynthesis and Function

描述（由申请人提供）：钼辅因子（Moco）由单核钼与一个独特的二噻吩基团的硫原子配位组成，该二噻吩基团是被取代的蝶呤衍生物钼钼苷的一部分。含有这种辅助因子的酶存在于所有三个生命系统发育王国中，并且在原核生物和包括人类在内的真核生物中，辅助因子的生物合成遵循相同的途径。人类Moco缺乏症是一种严重的遗传性疾病，表现为神经异常，并导致患者过早死亡。参与Moco生物合成的编码酶基因的第一个突变最近已经从患者身上得到了表征，突变的影响可以利用我们获得的大肠杆菌蛋白的结构来合理化和分析。此外，真细菌中参与二核苷酸形成的酶可能是合理设计抗菌抑制剂的合适候选者。本提案中概述的实验将通过结构和生化实验的结合进一步推进我们对这一保守途径所涉及的酶的理解。此外，对Moco生物合成过程中参与硫结合步骤的两种蛋白质的研究为泛素和相关蛋白质修饰剂的激活提供了重要的见解（SUMO, NEDD8）。本提案提出的具体目标可以概括如下：(1)研究该途径第一步所涉及的酶。具体而言，我们将确定MoaA蛋白的晶体结构，并进一步表征MoaC的活性位点和功能。(2) Moco二噻吩基形成酶的生化和结构研究。我们将研究MoaD与其结合伙伴MoeB和MoaE之间的相互作用，并表征钼钼苷合成酶和MoeB的活性位点。(3)通过MogA和MoeA的生化和结构表征，进一步表征参与金属结合步骤的蛋白质。(4) Moco生物合成过程中原核特异性步骤的研究。我们将研究MobA形成二核苷酸的机制，并确定病原菌中MobA蛋白的晶体结构，作为合理设计具有抗菌活性化合物的起点。

DESCRIPTION (provided by applicant): The molybdenum cofactor (Moco) consists of a mononuclear molybdenum coordinated to the sulfur atoms of a unique dithiolene group which is part of a substituted pterin derivative termed molybdopterin. Enzymes containing this cofactor are found in all three phylogenetic kingdoms of life, and biosynthesis of the cofactor follows the same pathway in prokaryotes and in eukaryotes including humans. Moco deficiency in humans is a severe inherited disease that manifests itself in neurological abnormalities and leads to premature death in he affected individuals. The first mutations in the genes encoding enzymes involved in Moco biosynthesis have recently been characterized from patients, and the effects of the mutations can be rationalized and analyzed using the structures of the E. coli proteins we have derived. In addition, the enzyme involved in dinucleotide formation in eubacteria might be a suitable candidate for the rational design of inhibitors that function as antimicrobial agents. The experiments outlined in this proposal will further advance our understanding of the enzymes involved in this conserved pathway through a combination of structural and biochemical experiments. Furthermore, studies of two proteins involved in the sulfur incorporation step during Moco biosynthesis provide important insights into the activation of ubiquitin and related protein modifiers (SUMO, NEDD8). The specific aims presented in this proposal can be summarized as follows: (1) Studies of the enzymes involved in the first step of this pathway. Specifically, we will determine the crystal structure of a MoaA protein, and further characterize the active site and function of MoaC. (2) Biochemical and structural studies of the enzymes involved in the formation of the dithiolene group of Moco. We will study the interactions between MoaD and its binding partners MoeB and MoaE and characterize the active sites of molybdopterin synthase and MoeB. (3) Further characterization of the proteins involved in the metal incorporation step through biochemical and structural characterization of MogA and MoeA. (4) Studies of the prokaryoto-specific steps during Moco biosynthesis. We will study the mechanism of dinucleotide formation by MobA and determine the crystal structures of MobA proteins from pathogenic bacteria as a starting point for the rational design of compounds with antimicrobial activity.