STRUCTURAL STUDIES OF REDOX ENZYMES AND MEDICALLY IMPORTANT SERINE PROTEASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这是一项为期两年的APS束流时间方案，用于生物大分子结构研究。它集中在四种氧化还原蛋白和几种哺乳动物蛋白酶突变体以及与天然或合成抑制剂的复合体上。 四聚体肌氨酸氧化酶(TSOX)中的氧化还原蛋白之一，TSOX是从嗜麦芽假单胞菌中分离出来的一种细菌黄素酶。它含有3种辅酶(FAD、FMN和NAD+)，由4个不同的亚基组成。TSOX催化肌氨酸氧化生成甲醛和过氧化氢。当有四氢叶酸存在时，肌氨酸氧化与5，10-亚甲基四氢叶酸的形成偶联。 另一种氧化还原酶是nikD，它催化肌腱链霉菌合成尼可霉素抗生素的早期步骤。它是一个45 kDa的单体，含有共价结合到半胱氨酸上的FAD。在不同的pH条件下，以封闭和开放的形式测定了NikD。 PCMH是从恶臭假单胞菌中分离到的一种异四聚体黄色素c，它含有一个2×59 kDa的黄素酶二聚体(FPSU)，含有共价结合的FAD和两个9 kDa的细胞色素亚基。它催化对甲酚氧化成对羟基苯甲醇，并将电子转移到细胞色素上。PCMH的1.85°结构和FPSU的1.3°结构是已知的。 氨蓝蛋白是一种9 kDa的蓝铜蛋白，它接受脱硝副球藻中甲胺脱氢酶的电子。氨基花青素的几个突变体的氧化还原电位、电子耦合和重组能与野生型蛋白有显著差异。 被研究的哺乳动物丝氨酸蛋白酶约为30 kDa，参与血栓形成和病理性炎症反应等过程。对这些酶的研究代表了实验室的一个主要重点。 这些蛋白质的几种突变形式以及各种酶-抑制物复合体的结构将被确定，以阐明它们的催化机制和模式。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This is a 2-year proposal for APS beam time for structural studies of biological macromolecules. It focuses on four redox proteins and on several mammalian protease mutants and complexes with natural or synthetic inhibitors. One of the redox proteins in tetrameric sarcosine oxidase (TSOX), a bacterial flavoenzyme isolated from Pseudomonas maltophilia. It contains three coenzymes (FAD, FMN and NAD+) and comprises 4 different subunits. TSOX catalyzes the oxidation of sarcosine to yield formaldehyde and peroxide. When tetrahydrofolate is present, sarcosine oxidation is coupled to the formation of 5,10-methylenetetrahydrofolate. Another redox enzyme is nikD, which catalyzes an early step in the biosynthetic pathway for nikkomycin antibiotics in Streptomyces tendae. It is a monomer of 45 kDa containing FAD bound covalently to cysteine. NikD has been determined in closed and open forms at different pH. PCMH is a heterotetrameric flavocytochrome c isolated from Pseudomonas putida that contains a 2x59-kDa flavoenzyme dimer (FPSU) containing covalently bound FAD and two 9 kDa cytochrome subunits. It catalyzes the oxidation of p-cresol to p-hydroxybenzyl alcohol and electron transfer to the cytochrome. The 1.85 ¿ structure of PCMH and the 1.3 ¿ structure of FPSU are known. Amicyanin is a blue copper protein of 9 kDa that accepts electrons from methylamine dehydrogenase in Paracoccus denitrificans. The redox potentials, electronic coupling and reorganization energies of several mutants of amicyanin show significant differences from the wild type protein. The mammalian serine proteases being studied are approximately 30 kDa and are involved in processes such as thrombosis and pathological inflammatory response. Research on these enzymes represents a major focus of the laboratory. The structures of several mutant forms of these proteins, as well as of various enzyme-inhibitor complexes will be determined in order to elucidate their catalytic mechanisms and modes of