AIM:To determine if and how a loop region in the peptide deformylase(PDF)of Chlamydia trachomatis regulates enzyme function. METHODS:Molecular dynamics simulation was used to study a structural model of the chlamydial...AIM:To determine if and how a loop region in the peptide deformylase(PDF)of Chlamydia trachomatis regulates enzyme function. METHODS: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. RESULTS: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 isresistant 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. CONCLUSION: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.展开更多
基金Supported by A grant from the National Institutes of Health (AI071954)to Fan H
文摘AIM:To determine if and how a loop region in the peptide deformylase(PDF)of Chlamydia trachomatis regulates enzyme function. METHODS: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. RESULTS: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 isresistant 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. CONCLUSION: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.
