REVERSIBLE ADP-RIBOSYLATION OF NITROGENASE

本项目的目标是了解 可逆ADP-核糖化的固氮酶还原酶。固氮酶 红色红螺菌固氮酶的还原酶蛋白 被ADP核糖化的arg101对氨或黑暗的反应灭活。 DRAT是负责失活的ADP-核糖基转移酶，而阻力是 通过去除ADP-核糖来激活脱氮酶还原酶的糖水解酶。 DRAT和阻力在单元格中是独立调节的，总体上 这个项目的目标是了解信号转导途径 调节DRAT和阻力。DRAT和Drag的结构信息将是 通过结晶学、序列模拟和特定部位的化学物质获得 酶的修饰。这些信息将被用来指导 DRAT和DRAT编码基因的突变，以及DRAT的突变形式， 将检测它们的活性和脱氮酶还原酶的活性 监管。NAD作为ADP核糖供体，与NAD结合的部位 将建立DRAT和脱氮酶还原酶。假设 将测试调节DRAT活性的NAD结合位点的占有率 体外和体内；紫外光亲和技术将被开发用于 活体测试。NAD与DRAT和脱氮酶还原酶的结合将是 量化的。将在体外寻找DRAT和阻力活动的效应物 化验。不受调控的DRAT和DRAT的突变形式将始终处于活动状态 在这些检测中作为对照。效应器将被隔离和识别。 腺苷二磷酸核糖基化是人类免疫缺陷的重要机制。 真核生物和原核生物的调控。一些致病菌 产生ADP-核糖化的毒素，使它们的真核细胞中的关键蛋白质靶标 主持人。

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.