High tolerance to mutations in a Chlamydia trachomatis peptide deformylase loop

To determine if and how a loop region in the peptide deformylase (PDF) of Chlamydia trachomatis regulates enzyme function.

Molecular dynamics simulation was used to study a structural model of the chlamydial PDF (cPDF) and predict the temperature factor per residue for the protein backbone atoms. Site-directed mutagenesis was performed to construct cPDF variants. Catalytic properties of the resulting variants were determined by an enzyme assay using formyl-Met-Ala-Ser as a substrate.

In silico analysis predicted a significant increase in atomic motion in the DGELV sequence (residues 68-72) of a loop region in a cPDF mutant, which is resistant to PDF inhibitors due to two amino acid substitutions near the active site, as compared to wild-type cPDF. The D68R and D68R/E70R cPDF variants demonstrated significantly increased catalytic efficiency. The E70R mutant showed only slightly decreased efficiency. Although deletion of residues 68-72 resulted in a nearly threefold loss in substrate binding, this deficiency was compensated for by increased catalytic efficiency.

Movement of the DGELV loop region is involved in a rate-limiting conformational change of the enzyme during catalysis. However, there is no stringent sequence requirement for this region for cPDF enzyme activity.