Structure, Function and Inhibition of [4Fe-4S] Proteins

描述（由申请人提供）：这项工作的目的是研究三种不同类型的金属蛋白的结构、作用机制和抑制作用，它们都利用[4Fe-4S]簇进行催化。催化的反应是还原性去羟基化，脱水和异构化，所有的蛋白质都是长期感兴趣的，因为它们是许多病原体生存所必需的，而不是被人类使用。这些蛋白是IspG和IspH，参与类异戊二烯生物合成中的还原性去羟基化；喹啉酸合成酶（NadA），参与NAD的生物合成；二羟基酸脱水酶（DHAD）和异丙酸异构酶（LeuCD），参与氨基酸的生物合成。在Aim 1中，第一个目标是研究在许多厌氧菌中发现的以前未被描述的一类IspHs，例如导致破伤风和肉毒杆菌中毒的细菌，以及在人类微生物组中发现的许多细菌。这些蛋白比大多数细菌的isph大2-3倍，含有IspH-RPS1（核糖体结合蛋白S1）融合，可能作为铁或氧传感器。第二个目标是通过使用快速冻冷EPR、量热法、定点诱变、M¿ssbauer、x射线晶体学和DFT来表征反应中间体，探索IspG和IspH的作用机制。第三个目标是确定3-domain IspG的结构并开发新的IspG抑制剂。目的2涉及对NadA的研究，NadA是一种载脂蛋白结构类似于IspH的酶，但其4Fe-4S簇的结构尚不清楚。目的是确定其结构和作用机制，并开发抑制剂作为药物先导（针对引起胃溃疡和胃癌的生物体）。第三个目标涉及两种[4Fe-4S]水合酶/异构酶（DHAD和LeuCD）。预测两者都包含两个结构域，并且DHAD， LeuCD， aconitase和延胡索酶A之间似乎存在结构相似性，我们将对这一假设进行验证。抑制剂已被报道为除草剂，阻断亮氨酸和其他支链氨基酸的生物合成，与LeuCD抑制剂一起使用的效果与L-亮氨酸相反，结核分枝杆菌LeuCD敲除被开发为结核病疫苗，使LeuCD和DHAD成为潜在的新药物靶点，在这里，我们将开发抑制剂，在细胞中活跃，与它们的[4Fe-4S]簇结合。

DESCRIPTION (provided by applicant): The objective of this work is to investigate the structure, mechanism of action, and inhibition of three different types of metalloprotein, all of which utilize [4Fe-4S] clusters in catalysis. The reactions catalyzed are reductive dehydroxylations, dehydrations and isomerizations, and all of the proteins are of long-term interest because they are essential for the survival of many pathogens and are not used by humans. The proteins are IspG and IspH, involved in reductive dehydroxylations in isoprenoid biosynthesis; quinolinate synthase (NadA), involved in NAD biosynthesis; and dihydroxyacid dehydratase (DHAD) and isopropylmalate isomerase (LeuCD), involved in amino-acid biosynthesis. In Aim 1 the first objective is to investigate a previously uncharacterized class of IspHs that are found in many anaerobic bacteria, such as those which cause tetanus and botulism, as well as many which are found in the human microbiome. These proteins are 2-3x larger than most bacterial IspHs and contain an IspH-RPS1 (ribosomal binding protein S1) fusion and might act as iron or oxygen sensors. The second objective is to probe the mechanisms of action of both IspG and IspH by using rapid freeze-quench EPR, calorimetry, site-directed mutagenesis, M¿ssbauer, X-ray crystallography, and DFT to characterize reaction intermediates. The third objective is to determine the structures of the 3-domain IspGs and develop novel IspG inhibitors. Aim 2 involves investigation of NadA, an enzyme whose apo-structure resembles that of IspH but whose structure with its 4Fe-4S cluster is unknown. The objective is to determine its structure and mechanism of action and to develop inhibitors as drug leads (against the organism that causes gastric ulcers and carcinoma). The third Aim involves two [4Fe-4S] hydratase/isomerases (DHAD and LeuCD). Both are predicted to contain two domains and it appears that there are structural similarities between DHAD, LeuCD, aconitase and fumarase A, a hypothesis we will test. Inhibitors have been reported as herbicides, blocking leucine and other branched chain amino-acid biosynthesis, an effect that with the LeuCD inhibitor is reversed with L- leucine, and M. tuberculosis leuCD knockouts are being developed as TB vaccines, making LeuCD and DHAD potential new drug targets and here, we will develop inhibitors, active in cells, that bind to their [4Fe-4S] clusters.