The Molybdenum Cofactor: Biosynthesis and Function

描述（由申请人提供）：钼辅因子（Moco）由与独特二硫杂环戊烯基团的硫原子配位的单核钼组成，该二硫杂环戊烯基团是被称为蝶呤的取代蝶呤衍生物的一部分。 含有这种辅因子的酶存在于所有三个系统发育的生命王国中，辅因子的生物合成在原核生物和包括人类在内的真核生物中遵循相同的途径。 人类的莫科缺乏症是一种严重的遗传性疾病，表现为神经系统异常，并导致受影响个体的过早死亡。 最近已经从患者中表征了参与Moco生物合成的编码酶的基因中的第一个突变，并且可以使用E.大肠杆菌的蛋白质。 此外，真细菌中参与二核苷酸形成的酶可能是合理设计具有抗菌作用的抑制剂的合适候选物。 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中所涉及的酶的理解。 此外，对参与Moco生物合成过程中硫掺入步骤的两种蛋白质的研究为泛素和相关蛋白质修饰剂的活化提供了重要的见解（SUMO，NEDD 8）。 本研究的具体目标可以概括如下：（1）研究该途径第一步的酶。 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)对参与莫科二硫纶基团形成的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，并表征蝶呤合酶和MoeB的活性位点。(3)通过莫加和MoeA的生化和结构表征进一步表征金属掺入步骤中涉及的蛋白质。(4)莫科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MobA蛋白的晶体结构，作为合理设计具有抗微生物活性的化合物的起点。

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.