Structural and Mechanistic Studies of 10-Formyltetrahydrofolate Synthetase

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Lukasz LebiodaMCB 987360610-Formyltetrahydrofolate synthetase (FTHFS) catalyzes the reversibleformylation of tetrahydrofolate using the energy of ATP hydrolysis to drivethe reaction. All known FTHFSs are homologous; however, in many eukaryotesFTHFS is a part of trifunctional C-1-tetrahydrofolate synthase while inbacteria it is a separate enzyme. The mechanism of FTHFS has been studiedfor almost three decades but in the absence of information about itsthree-dimensional structure the progress has been limited. Of particularinterest is the role of monovalent cations in catalysis, the comparison ofthe folate-binding site with those present in other enzymes. The enzyme ofinterest is from thermophilic bacteria and the structural origins ofthermostability will be analyzed.This study is based on previous studies with 10-Formyltetrahydrofolatesynthetase from Clostridium thermoaceticum. Earlier work determined thecDNA and amino acid sequence, expressed the enzyme in E. coli, purified andcrystallized the recombinant native and selenomethionine forms of theenzyme. Multi-wavelength anomalous dispersion (MAD) data were collected to2.5 A resolution from the Se-Met crystals. Positions of anomalousscatterers were determined using direct methods and the results used tosolve the phase problem. A model of the molecule was built and optimizedusing crystallographic refinement. Further studies will be directed towardsthe determination of the structures of the enzyme in the form of complexeswith the substrates/catalytic intermediates, and inhibitors. Based onstructural data, the catalytic role of side chains present in the activesite will be elucidated. The hypotheses so generated will be tested usingsite-directed mutagenesis.