STRUCT STUD OF ENZYMES INVOLVED IN COFACTOR BIOSYNTHESIS AND NUCLEOSIDE SALVAGE

这个子项目是众多研究子项目之一

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. Our research focuses on the three-dimensional structures of enzymes, with emphasis on drug design, protein engineering, determination of enzyme mechanism and protein evolution. In the area of purine and pyrimidine nucleosides, we used SeMet MAD phasing to determine the structure of two different crystal forms of AMP nucleosidase, which plays a key role in the purine salvage pathway. In addition, structures of inhibitor bound AMP nucleosidase allow for greater understanding of the biological functions and enzymatic mechanism of the protein family. Uridine phosphorylase (UDP) is an E. coli PNP homolog with specificity for pyrimidines versus purines. Co-crystals of UDP with 5-fluorouridine and with uracil and ribose-1-phosphate clearly show the important active site residues and also show the first structurally characterized oxocarbenium intermediate. SeMet SAD phasing was used to determine the structure of the essential multifunctional enzyme PurL from Salmonella typhimurium, which consists of three domains homologous to three separate proteins in other organisms. This structure will allow for insights into protein/protein interactions and evolution. The structure of Bacillus subtilis PurS was solved by MR and plays a key role in purine metabolism. SAD data was collected for small PurL from Thermatoga maritima and the structure solution is progressing. Acetobacter aceti PurE was solved using MR and provides a deeper understanding into the mechanism of PurE and general protein acid stability. SeMet SAD phasing was used to solve the structure of AirS kinase. This enzyme functions at the interface of the purine and thiamin biosynthetic pathways. The structure will provide insights into both pathways and into the evolution of the ribokinase superfamily. In the area of polyamine biosynthesis, we used MR to determine the structures of two mutants of pyruvoyl-dependent arginine decarboxylase (PvlArgDC). The structures may aid in designing inhibitors that interfere with the autoprocessing of human S-adenosylmethionine decarboxylase (AdoMetDC). In the area of cofactor biosynthesis, we used MR to determine the structure of human phosphopantothenoylcysteine decarboxylase (PPC decarboxylase), which catalyzes the second step in the synthesis of Coenzyme A from phosphopantothenate. This enzyme is a potential drug design target. The structure of ThiSG has been determined by SAD phasing and various mutants are being sought to provide insights into mechanisms.