REVERSIBLE ADP-RIBOSYLATION OF NITROGENASE

描述（改编自申请人的摘要）：ADP-核糖基化是一种 真核生物酶活性调节重要机制， 原核生物 这种蛋白质修饰在许多方面起着重要作用。 人类疾病。 固氮酶的二固氮酶还原酶蛋白 来自光合细菌Rhodocellum rubrum的酶复合物， 由arg-101的可逆ADP-核糖基化调节。 ADP-核糖基化 在体内发生，以响应黑暗或铵，并被催化， 二氮酶还原酶ADP-核糖基转移酶（DRAT）。 Mn依赖 ADP-核糖的去除是由二氮酶还原酶活化催化的， 糖水解酶（DRAG）。 DRAT和DRAG活性在体内都受到调节 该提案的目的是确定体内 调节这两种酶。 纯化的DRAT和DRAG完全活跃， 因此，这种调节被认为是通过抑制它们的 活动 一个涉及DRAT和DRAG活动的监管的模型， 形成含有DRAT：DRAG的蛋白质：蛋白质复合物，或 DRAT：二氮酶还原酶已被开发。 具体目标是 建议表征DRAT与二氮酶的复合物 还原酶和DRAT与DRAG的反应。 控制形成的效应器 将寻找和确定复合体。 证据 DRAT：二氮酶还原酶复合物已经使用化学方法获得。 交联剂EDC。 DRAT的形成：二氮酶还原酶 复合物需要NAD的存在，并由ADP刺激。 初步 证据还表明存在DRAT：DRAG复合物。 模型 要求小分子效应器参与这些复合物， 该提案的目标是隔离效应器，识别它们并证明 效应物浓度的变化与体内 二氮酶还原酶的ADP-核糖基化。 生理学实验 暗示了dratTG基因下游orf的产物， DRAT和DRAG的调节以及从一个 过表达菌株是本项目的目标。 将特别注意 考虑到ORF产物参与任何蛋白质的能力 复杂. DRAG的Mn结合位点将被表征，并且 Mn在刺激/抑制DRAG与其他蛋白质的任何复合物中的作用将是可能的。 研究了 Mn构成双核团簇的假说 将对DRAG进行测试。

DESCRIPTION (Adapted from the applicant's abstract): ADP-ribosylation is an important mechanism of regulation of enzyme activity in eucaryotes and procaryotes. This protein modification plays an important role in a number of human diseases. The dinitrogenase reductase protein of the nitrogenase enzyme complex from the photosynthetic bacterium Rhodospirillum rubrum is regulated by reversible ADP-ribosylation of arg-101. ADP-ribosylation occurs in vivo in response to darkness or ammonium and is catalyzed by the dinitrogenase reductase ADP-ribosyltransferase (DRAT). The Mn-dependent removal of ADP-ribose is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG). Both DRAT and DRAG activities are regulated in vivo and the goal of this proposal is to determine the mechanism(s) of in vivo regulation of these two enzymes. Purified DRAT and DRAG are fully active, and thus the regulation is thought to occur by inhibition of their activities. A model that involves regulation of DRAT and DRAG activities by formation of protein:protein complexes that contain DRAT:DRAG or DRAT:dinitrogenase reductase has been developed. The specific aims of this proposal are to characterize the complexes of DRAT with dinitrogenase reductase and of DRAT with DRAG. Effectors that control the formation of complexes will be sought and identified. Evidence for the DRAT:dinitrogenase reductase complex has been obtained using chemical crosslinking agent EDC. Formation of the DRAT:dinitrogenase reductase complex requires the presence of NAD and is stimulated by ADP. Preliminary evidence also suggests the existence of the DRAT:DRAG complex. The model calls for small molecule effectors to be involved in these complexes and a goal of the proposal is to isolate effectors, identify them and demonstrate changes in concentration of the effectors in concert with in vivo ADP-ribosylation of dinitrogenase reductase. Physiological experiments have implicated the product of an orf downstream of the dratTG genes in the regulation of DRAT and DRAG and the purification of the orf product from an overexpressing strain is a goal of this project. Particular attention will be paid to the ability of the orf product to participate in any protein complex. The Mn-binding site of DRAG will be characterized and the role of Mn in stimulating/inhibiting any complex of DRAG with other proteins will be investigated. The hypothesis that Mn constitutes a binuclear cluster in DRAG will be tested.