STRUCTURAL STUDIES OF TSCP AND ITS MUTANTS

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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. The thy1 gene of Thermotoga maritima encodes a thymidylate synthase complementing protein (TSCP), TM0449. TSCPs have been implicated in cell survival in the absence of external sources of thymidylate. In general, TSCP?s complement the activity of thymidylate synthase (TS). Thymidylate synthesis is the terminal step in the sole de novo synthetic pathway to dTMP. Consequently, TS inhibition stops DNA production, arresting the cell cycle and eventually leading to cell death. The TSCP family share no sequence of structural homology to classical TS. Although extremely rare in Eukaryotes the TSCP gene is widely distributed within the bacterial domain of life. Many members of the TSCP family are human pathogenic bacteria. In continuation with our structural study of TM0449, we have studied the structures of 12 muitants that are aimed at understanding the functional aspects of the enzyme. The structural study of the three double mutants (F158G-W160A,F158A-W160A, F158A-F160Q) with substrates and cofactors helps to understand the mechanistic aspects of the enzyme catalysis. The current focus of the project is to explore the mechanism of TSCP using enzymes from pathogens causing Anthrax, Typhus, and Tuberculosis. We have just started the crystallization of the TSCP enzyme from Rickettsia. The structures of enzymes from different organisms will help us understand the important functional groups and the possibility of the design of an antibiotic-drug. Our structural study to trap the intermediates under anaerobic conditions is also in progress. The initial structures from two of these complexes prepapred under anerobic conditions show that the flavine ring of the FAD is disordered in the FAD-enzyme complex as well as the FAD-enzyme-dUMP complex. Therefore, we are now employing better chemical procedures to trap the folate in the active site. The new collaboration with Professor Amnon Kohen?s group provides lot of mechanistic insights to the complex synthesis, anaerobic experiments, and crystallization.