STRUCTURAL STUDIES OF TM

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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 now collected diffraction data for around 12 mutants of the enzyme. These mutants 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 study is focused on preparing the complexes and crystallizing them in an anaerobic atmosphere. We have grown crystals of two mutants under anaerobic conditions and the structural study is in progress. The rapid conversion of the reduced FAD to the oxidized form in the presence of small amounts of oxygen is a major problem in crystallizing complexes with the reduced form of FAD. There are no structures available with the reduced form of FAD bound in the active site. The structural information derived from the complexes prepared under anaerobic conditions will be valuable for the mechanistic study of the TSCP catalysis.