The long-term objectives of this project are to understand the molecular mechanism of peptide deformylase, a small zinc-containing enzyme (169 amino acids in Escherichia coli), and design deformylase inhibitors and deformylase activated prodrugs as potential antibacterial agents. Ribosomal protein synthesis universally initiates with a methionine residue. In prokaryotes and the organelles of eukaryotes, the methionyl moiety attached to initiator tRNA is N-formylated prior to its incorporation. Following translational initiation, the N-formyl group is removed from the vast majority of bacterial proteins, a process catalyzed by the peptide deformylase. It has been demonstrated that proper deformylation of nascent polypeptides is essential for bacterial survival, as deletion of the deformylase gene (def) is lethal in E. coli. However, this deformylation process is apparently dispensable in eukaryotes. Therefore, deformylase appears to be an ideal target for developing broad-spectrum antibiotics, since formylation is a conserved feature in all eubacteria.
Specific aims i nclude: 1) Determination of the substrate specificity of deformylase using synthetic substrate analogues and combinatorial library screening; 2) Determination of catalytic mechanism using isotope exchange and chemical trapping experiments; 3) Determination of its 3-D structure by X-ray crystallography; 4) Determination of the function of its Zn2+ ion and identification of active-site residues by spectroscopic methods and site-directed mutagenesis; and 5) Design and synthesis of deformylase inhibitors and prodrugs that could be activated by deformylase action in vivo.
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