REVERSIBLE ADP-RIBOSYLATION OF NITROGENASE

本项目的目标是了解 二氮酶还原酶通过可逆的ADP-核糖基化。二氮酶 红色红螺菌固氮酶的还原酶蛋白是 通过响应铵或黑暗的arg 101的ADP β-核糖基化失活。 DRAT是负责失活的ADP-核糖基转移酶，DRAG是负责失活的ADP-核糖基转移酶。 通过除去ADP-核糖来激活二氮酶还原酶的糖水解酶。 DRAT和DRAG在细胞中都是独立调节的， 该项目的目标是了解信号转导途径， 调节DRAT和DRAG。DRAT和DRAG的结构信息将 通过晶体学、序列建模和位点特异性化学分析获得 酶的修饰。这些信息将用于指导 诱变编码DRAT和DRAG的基因，以及DRAT的突变形式， DRAG和二固氮酶还原酶的活性将被测试， 调控NAD充当ADP-核糖供体，并且NAD结合至ADP-核糖供体的位点是NAD结合至ADP-核糖的位点。 将建立DRAT和二氮还原酶。的假设 NAD结合位点的占据调节DRAT活性将在 体外和体内; UV光亲和技术将被开发用于体内 体内试验NAD与DRAT和二氮酶还原酶的结合将是 量化。DRAT和DRAG活性的效应物将使用体外 测定。不受调节的DRAT和DRAG的突变形式“总是活跃的”， 在这些试验中用作对照。将分离并鉴定效应物。 ADP-核糖基化越来越被认为是一种重要的机制， 真核生物和原核生物的调控。一些致病菌 产生毒素，ADP-核糖基化关键蛋白质的目标，在他们的真核细胞 hosts.

The goal of this project is to understand the regulation of dinitrogenase reductase by reversible ADP-ribosylation. The dinitrogenase reductase protein of the nitrogenase enzyme in Rhodospirillum rubrum is inactivated by ADP{-ribosylation of arg101 in response to ammonium or darkness. DRAT is the ADP-ribosyltransferase responsible for inactivation and DRAG is the glycohydrolase that activates dinitrogenase reductase by removing ADP-ribose. Both DRAT and DRAG are independently regulated in the cell, and the overall goal of this project is to understand the signal transduction pathways that regulate DRAT and DRAG. Structural information for both DRAT and DRAG will be obtained by crystallography, sequence modeling and site-specific chemical modification of the enzymes. This information will be used to direct mutagenesis of genes encoding DRAT and DRAG, and the mutant forms of DRAT, DRAG, and dinitrogenase reductase will be tested for their activities and regulation. NAD serves as the ADP-ribose donor, and the site of NAD binding to DRAT and dinitrogenase reductase will be established. The hypothesis that occupancy of the NAD binding sites regulates DRAT activity will be tested in vitro and in vivo; UV photoaffinity techniques will be developed for the in vivo tests. Binding of NAD to DRAT and to dinitrogenase reductase will be quantified. Effectors of DRAT and DRAG activities will be sought using in vitro assays. Mutant forms of DRAT and DRAG that are unregulated "always active" will serve as controls in these assays. Effectors will be isolated and identified. ADP-ribosylation is increasingly recognized as an important mechanism of regulation in eucaryotes and procaryotes. A number of pathogenic bacteria produce toxins that ADP-ribosylate crucial protein targets in their eucaryotic hosts.