STRUCTURAL STUDIES OF REDOX ENZYMES AND MEDICALLY IMPORTANT SERINE PROTEASES

点击翻译按钮获取中文摘要

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