BIOSYNTHESES OF FEMO-CO AND FEV-CO OF NITROGENASE

钼的铁钼辅因子（FeMo-co）的生物合成 固氮酶和钒的铁钒辅因子（FeV-co） 将研究固氮酶。 这些辅因子作为活性物质， 固氮酶中进行ATP依赖的N2还原的位点 转化为铵，并提供蛋白质、核酸合成所需的N- 酸和其他含氮化合物。 FeMo-co是最好的 已知的辅因子，由MoFe 7S 8 - 9高柠檬酸盐组成; FeMo-Co的结构最近已被确定。 铁钒 辅因子被认为具有与FeMo-co非常相似的结构。 本项目的具体目标是：1）确定结构 以及最近确定的FeMo-Co前体的作用。 这种化合物是由具有活性nifB基因的细胞产生的，称为 NifB-co. NifB-co是合成FeMo-co和FeV-co所必需的。 有限 2)开发NifB-co的体外合成测定。 为了确定nifNE和vnfN基因产物在FeMo-co中的作用， 和FeV-co合成。 我们的假设是NifNE和 VnfNE充当其上组装FeMo-co和FeV-co的支架 4)确定nifH和vnfH基因产物的作用 （二氮酶还原酶）在FeMo-co和FeV-co合成中的作用。 我们 假设这些基因产物在制备NifNe和NifNe中起作用， nifH和vnfH基因产物也可能是NifB-co结合的蛋白质。 在指定添加到NifB-co中的金属（Mo或V）中起作用。 5)确定合成以下化合物所需的任何其他组分： 有强有力的证据表明，FeMo-co合成需要 除了NifB-co，NifNE蛋白，NifH蛋白， 高柠檬酸盐、过氧化氢、ATP和还原剂。 体外FeMo-co 将使用合成分析来鉴定这些因素，并将使用 以遵循因子的纯化。 的途径 要阐明这种生物合成途径， 涉及的产品和中间体，表征和识别它们， 从这些信息中推断出路径。 对某些方面的要求 预计这些步骤是复杂的。 许多组分和 该途径的中间体是氧不稳定的，因此大多数的 分离和表征将使用厌氧 技术. 虽然固氮的能力仅限于多种 在原核生物中，固定的氮是最终的氮源。 地球上大部分生命的起源。 固氮酶含有络合金属 集群和这些集群的生物合成是这个的目标， 项目 Mo和Fe以及这些团簇的重要组分， 元素对所有生物体的健康至关重要。 的理解 钼、铁和钒的加工将在本项目中获得 也适用于其他系统。

The biosynthesis of the iron-molybdenum cofactor (FeMo-co) of molybdenum nitrogenase and the iron-vanadium cofactor (FeV-co) of the vanadium nitrogenase will be investigated. These cofactors serve as the active sites in nitrogenases which carry out the ATP-dependent reduction of N2 to ammonium and provide the N-needed for synthesis of proteins, nucleic acids and other nitrogenous compounds in the cell. FeMo-co is the bette understood of the cofactors and consists of MoFe7S8-9homocitrate; the structure of FeMo-co has recently been determined. The iron-vanadium cofactor is thought to have a structure that is very similar to FeMo-co. The specific goals of this project are: 1) To determine the structure and the role of a recently identified precursor to FeMo-co. This FeS compound is produced by cells with an active nifB gene and is called NifB-co. NifB-co is required for the synthesis of both FeMo-co and FeV- co. 2) To develop an assay for the in vitro synthesis of NifB-co. 3) To determine the role(s) of the nifNE and vnfN gene products in FeMo-co and FeV-co synthesis, respectively. Our hypothesis is that NifNE and VnfNE serve as the scaffolds upon which FeMo-co and FeV-co are assembled 4) To determine the role(s) of the nifH and vnfH gene products (dinitrogenase reductase) in FeMo-co and FeV-co syntheses. Our hypothesis is that these gene products play a role in preparing NifNe an VnfNE proteins to bind NifB-co. The nifH and vnfH gene products may als play a role in specifying the metal (Mo or V) that is added to NifB-co. 5) To identify any additional components required for the synthesis of FeMo-co. There is strong evidence that FeMo-co synthesis requires components in addition to NifB-co, the NifNE protein, the NifH protein, homocitrate, molybdate, ATP and reductant. The in vitro FeMo-co synthesis assay will be used to identify those factors and will be used to follow the purification of the factors. The approach to the elucidation of this biosynthetic pathway will be to isolate the gene products and intermediates involved, characterize and identify them and, from this information, to deduce the pathway. Requirements for some of the steps are expected to be complex. Many of the components and intermediates of this pathway are oxygen-labile, and thus most of the isolations and characterizations will be performed using anaerobic techniques. While the ability to fix nitrogen is limited to a diverse set of procaryotic organisms, the nitrogen fixed is the ultimate N sourc for much of the life on earth. Nitrogenases contain complex metal clusters and the biosyntheses of these clusters is the goal of this project. Mo and Fe and important components of these clusters and these elements are essential for health of all organisms. The understanding of Mo, Fe and V processing that is gained in this project will be applicable to other systems as well.