Structural Studies on Nitric Oxide Synthase

该提案的中心是血红素蛋白的结构与功能关系，特别强调 血红素巯基化酶、细胞色素P450和一氧化氮合酶（NOS）。P450在药物治疗中发挥重要作用 解毒，类固醇生物合成，并在各种天然有机化合物的氧化同化， 微生物的对于P450，我们目前的工作重点是P450与其 氧化还原伙伴。P450-伴侣复合物只有3种晶体结构，其中之一， P450 cam和它的氧化还原伴侣（一种叫做Pdx的铁氧还蛋白）之间形成的，表明Pdx诱导了一个大的 P450 cam结构变化，我们假设是质子耦合电子转移所必需的。P450凸轮是 对Pdx非常特异，没有其他铁氧还蛋白或相关蛋白可以支持P450 cam催化。现在的目标 是问这个属性是否是P450的一个更普遍的特征，如果不是，为什么不是。是什么 生物学上的优势来达到如此高的控制水平？为了探讨这些问题，我们计划深入研究其他 P450氧化还原伴侣，以更好地了解氧化还原伴侣结合如何影响质子耦合电子 转移反应我们对P450的研究也扩展到哺乳动物P450，尤其是P4503 A4， P450在药物代谢中的重要作用。我们的目标是开发一种药效团 使用新的抑制剂探测P4503 A4活性的动力学和适应性。这将提供 P4503 A4中药物相互作用和变构机制的重要信息。有了NOS，我们的努力 基于结构的抑制剂/药物设计。众所周知，NO的过量产生与以下因素有关： 一些病理条件，我们目前正在关注神经退行性疾病，黑色素瘤， 和致病菌。在这3种情况下，我们知道通过阻断NOS来抑制NO的产生， 潜在的治疗益处。我们现在正在使用广泛的方法来开发选择性的 3种潜在靶点的NOS抑制剂。最后一个领域是血红素运输的结构生物学， 细菌病原体某些细菌病原体必须通过摄取游离血红素， 血红素降解和铁释放。这需要一个涉及几种蛋白质的复杂运输系统 其中许多是膜结合的。我们的目标是研究血红素运输的结构生物学， 特别是为了更好地理解血红素成功传递所需的许多蛋白质-蛋白质相互作用 从血红蛋白到细菌细胞质。

This proposal centers on structure-function relationships in heme proteins with a special emphasis on the heme thiolate enzymes cytochrome P450 and nitric oxide synthase (NOS). P450s play critical roles in drug detoxification, steroid biosynthesis, and in the oxidative assimilation of various natural organic compounds by microorganisms. With P450s our efforts currently are focused on the interaction between P450s and their redox partners. There are only 3 crystal structures of a P450-partner complex and one of these, the complex formed between P450cam and its redox partner (a ferredoxin called Pdx), shows that Pdx induces a large structural change in P450cam that we hypothesize is required for proton coupled electron transfer. P450cam is quite specific for Pdx and no other ferredoxin or related protein can support P450cam catalysis. The goal now is to ask whether or not this property is a more general feature of P450s and if not, why not. What is the biological advantage for such a high level of control? To probe these questions we plan to study in depth other P450 redox partners to better understand how redox partner binding effects the proton coupled electron transfer reaction. Our studies on P450s also extend to mammalian P450s and especially P4503A4, the most abundant and important human P450 for drug metabolism. Our goal here is to develop a pharmacophore using novel inhibitors that probe the dynamics and adaptability of the P4503A4 active. This will provide important information on drug-drug interactions and allosteric mechanisms in P4503A4. With NOS, our efforts center on structure-based inhibitor/drug design. The overproduction of NO is well known to be associated with a number of pathological conditions and we currently are focusing on neurodegenerative diseases, melanoma, and pathogenic bacteria. In each of these 3 cases we know that inhibiting NO production by blocking NOS has potential therapeutic benefits. We now are using a broad range of approaches toward developing selective NOS inhibitors for each of the 3 potential targets. One final area is the structural biology of heme transport in bacterial pathogens. Certain bacterial pathogens must acquire host iron by taking up free heme followed by heme degradation and release of iron. This requires a complex transport system involving several proteins many of which are membrane bound. The goal here is to work out the structural biology of heme transport and especially to better understand the many protein-protein interactions required for successful delivery of heme from hemoglobin to the bacterial cytoplasm.