Poster: Enzymology

Abs # 246: Peptide deformylase: Site-directed mutation directed towards engineering inhibitor resistance

Presenter:	Dirk, Lynnette M.A., ldirk@uky.edu
Authors	Williams, Mark A. (A)   Houtz, Robert L. (A) (B)  Dirk, Lynnette M.A. (A) (B)
Affiliations:	(A): Department of Horticulture, University of Kentucky (B): Plant Physiology/Biochemistry /Molecular Biology Program

The initiating residue of protein translation in chloroplasts and mitochondria is N-formyl-methionine which is subsequently deformylated in the vast majority of proteins. Two chloroplast targeted peptide deformylases (DEFs), the enzyme responsible for N-formyl group removal, represent ideal molecular targets for the development of a new class of broad-spectrum herbicides. Although resistance to DEF inhibitors has been reported on seven occasions in bacteria, the mechanism of resistance involved an altered form of DEF in only a single organism. Resistance to DEF inhibitors in Streptococcus pneumoniae (Margolis et al., 2001) developed in three separate instances, two of which were the same A123D mutation just upstream of the conserved DEF Motif 2 (EGCL), which includes the metal ligating cysteine. The last instance (Q172K) involved a highly conserved glutamine of Motif 3 (HExxH), which includes the two metal ligating histidines. This mutation and one case of the A123D mutations resulted in a 4 fold increase in resistance to the DEF inhibitor actinonin. Corresponding mutations will be constructed for both AtDEF1 and 2 using site-directed mutagenesis. These AtDEF partial cDNAs will be in inducible vectors used to produce protein in E. coli. The activities of purified proteins will be assessed by in vitro-enzyme linked assays in the presence and absence of actinonin. Thus, these tests will give direct indications of a protein’s resistance to actinonin.