All nascent polypeptides synthesized in bacteria, mitochondria, or chloroplastsstart with a N-formylmethionine. Peptide deformylase (PDF) is a mononuclear metal ionprotein that is responsible for removing the N-formyl group of nascent proteins found inbacteria and chloroplasts in order for them to become mature proteins. It is possible, asseen from the literature with actinonin, to chelate the enzyme's metal ion and inhibit thefunction of protein production essentially resulting in death of the bacteria, or plant. Thisstudy examines the active site of Arabidopsis thaliana (At) types of PDF (AtDEF1 andAtDEF2, respectively) as well as bacterial DEF2 using sequence alignments andcomputational modeling. This work also investigates the biological efficacy of designingand synthesizing inhibitors that mimic actinonin or the D1 substrate that will halt, orseverely retard, the activity of the PDF enzyme in vitro and in vivo. Through thisresearch, we were able to determine specific residues that were conserved amongst theplant DEF2 sequences that were present less than 20% of the time in plant DEF1 andbacteria DEF2. This data allowed us to hypothesize plant DEF2's substrate specificity aswell as a possible design that is selective towards plants and not bacteria. Also, based onpreliminary results, the novel thiol-actinonin chimera that was synthesized showedinhibition activity of AtDEF2 during in vitro enzyme assays.