Kiet T. Nguyen

Crystals of Peptide Deformylase from Plasmodium falciparum Reveal Critical Characteristics of the Active Site for Drug Design

Peptide deformylase catalyzes the deformylation reaction of the amino terminal fMet residue of newly synthesized proteins in bacteria, and most likely in Plasmodium falciparum, and has therefore been identified as a potential antibacterial and antimalarial drug target. The structure of P. falciparum peptide deformylase, determined at 2.8 A resolution with ten subunits per asymmetric unit, is similar to the bacterial enzyme with the residues involved in catalysis, the position of the bound metal ion, and a catalytically important water structurally conserved between the two enzymes. However, critical differences in the substrate binding region explain the poor affinity of E. coli deformylase inhibitors and substrates toward the Plasmodium enzyme. The Plasmodium structure serves as a guide for designing novel antimalarials.

An improved crystal form of Plasmodium falciparum peptide deformylase

An altered version of peptide deformylase from Plasmodium falciparum (PfPDF), the organism that causes the most devastating form of malaria, has been cocrystallized with a synthesized inhibitor that has submicromolar affinity for its target protein. The structure is solved at 2.2 Å resolution, an improvement over the 2.8 Å resolution achieved during the structural determination of unliganded PfPDF. This represents the successful outcome of modifying the protein construct in order to overcome adverse crystal contacts and other problems encountered in the study of unliganded PfPDF. Two molecules of PfPDF are found in the asymmetric unit of the current structure. The active site of each monomer of PfPDF is occupied by a proteolyzed fragment of the tripeptide‐like inhibitor. Unexpectedly, each PfPDF subunit is associated with two nearly complete molecules of the inhibitor, found at a protein–protein interface. This is the first structure of a eukaryotic PDF protein, a potential drug target, in complex with a ligand.

High-throughput screening of peptide deformylase inhibitors.

The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the dipeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories.