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Phylogenomic and Biochemical Characterization of Three Legionella pneumophila Polypeptide Deformylases

Microbiology,¹ Enzymology and Mechanistic Pharmacology,² Gene Expression and Protein Biochemistry,³ Medicinal Chemistry,⁴ Bioinformatics, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, Pennsylvania 19426⁵

Received 14 June 2005/ Accepted 26 April 2006

Legionella pneumophila is a gram-negative facultative intracellular human pathogen that can cause fatal Legionnaires' disease. Polypeptide deformylase (PDF) is a novel broad-spectrum antibacterial target, and reports of inhibitors of PDF with potent activities against L. pneumophila have been published previously. Here, we report the identification of not one but three putative pdf genes, pdfA, pdfB, and pdfC, in the complete genome sequences of three strains of L. pneumophila. Phylogenetic analysis showed that L. pneumophila PdfA is most closely related to the commonly known -proteobacterial PDFs encoded by the gene def. PdfB and PdfC are more divergent and do not cluster with any specific bacterial or eukaryotic PDF. All three putative pdf genes from L. pneumophila strain Philadelphia 1 have been cloned, and their encoded products have been overexpressed in Escherichia coli and purified. Enzymatic characterization shows that the purified PDFs with Ni²⁺ substituted are catalytically active and able to remove the N-formyl group from several synthetic polypeptides, although they appear to have different substrate specificities. Surprisingly, while PdfA and PdfB with Zn²⁺ substituted are much less active than the Ni²⁺ forms of each enzyme, PdfC with Zn²⁺ substituted was as active as the Ni²⁺ form for the fMA substrate and exhibited substrate specificity different from that of Ni²⁺ PdfC. Furthermore, the catalytic activities of these enzymes are potently inhibited by a known small-molecule PDF inhibitor, BB-3497, which also inhibits the extracellular growth of L. pneumophila. These results indicate that even though L. pneumophila has three PDFs, they can be effectively inhibited by PDF inhibitors which can, therefore, have potent anti-L. pneumophila activity.