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. Dihydrofolate reductases (DHFRs) catalyze the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate. DHFR's catalytic activity is necessary for cellular metabolism and division due to its requirement in the production of thymidylate, a precursor of thymine. DHFRs remain major targets for anticancer and antimicrobial drug discovery programs, however, key enzymic structure/function questions abound. Essentially, there is no consensus on the origin and path of proton donation in DHFR's reduction mechanism. We have previously collected data on several liganded forms of E. coli DHFR including DHFR/methotrexate (MTX) complex crystals to 1.0 and DHFR/folate (FOL) to 1.1 . At the resolution available from the MTX and FOL complexes, difference electron density for hydrogen atoms at the active site has become 'visible.' We have now crystallized three more ligand-bound forms of DHFR: one with MTX and NADPH bound, one with a newly characterized inhibitor called Lee568 and NADPH bound, and one a binary complex with Lee568 only bound. We have tested the MTX/NADPH crystal for X-ray diffraction on our in-house source and reflections can be measured to 1.7 resolution. Ternary structures of inhibitor/cofactor complexes at high resolution (beyond 1.2 ) will provide not only structural details but mechanistic insights into cofactor positioning and the hydride transfer step it initiates to complete one catalytic step. Further work is also required on Rnr1 cocrystals to further our understanding of its ligand binding surfaces and its catalytic mechanism. Rnr1 is an absolutely required enzyme for the de novo synthesis of deoxyribonucleotide diphosphates (dNDPs) from NDPs. Previously, our lab has solved the structure of apo and ligand-bound forms of Rnr1 using diffraction data collected at IMCA-CAT and BioCARS stations. We now have several new forms of Rnr1 crystals, some bound to effector nucleotides and some bound to peptides derived from proteins it binds.
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