Inhibitors of Pantothenate Kinase: Novel Antibiotics for Staphylococcal Infections

Abstract

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A biosynthetic pathway. Here we report the identification of the Staphylococcus aureus coaA gene and characterization of the enzyme. We have also identified a series of low-molecular-weight compounds which are effective inhibitors of S. aureus CoaA.Increasing reports of antibiotic resistance involving opportunistic gram-positive pathogens, including methicillin-resistant Staphylococcus aureus, have emphasized the critical need for the development of antimicrobial compounds with novel modes of action. Coenzyme A (CoA), an essential cofactor for maintaining life, is used in a multitude of biochemical reactions. In most bacteria, CoA is synthesized from pantothenic acid (vitamin B 5 ) in 5 steps (5), with the first step being the phosphorylation of pantothenate by pantothenate kinase (CoaA). Although this pathway also exists in eukaryotes, in most cases there is no sequence homology between the prokaryotic and eukaryotic CoA biosynthetic enzymes (7,9,12,18,24,27). Thus, there is the potential for developing highly specific inhibitors of bacterial CoA enyzmes.Unlike the case for other biosynthetic pathways of bacteria, the genes involved in CoA biosynthesis are not organized as operons. This has delayed the identification of the enzymes responsible for CoA synthesis, even though the intermediate chemical steps have been known since the 1960s (1). With the recent identification of the Escherichia coli genes encoding the enzymes CoaBC and CoaE, the entire pathway is now known for this organism (9,10,13,19,21). Interestingly, the gene coaA, which encodes the first enzyme in the pathway, has no homolog in the complete genome sequences of the S. aureus strains Mu50 and N315 (11).Cloning and purification of S. aureus CoaA. Initially, the coaA gene sequences in S. aureus strains Mu50 and N15 (GenBank accession numbers BA000017 and BA000018, respectively) were identified through searches of the ERGO comparative genomic database (previously WIT) (http://ergo .integratedgenomics.com/ERGO/) (8). We cloned the S. aureus RN4220 coaA gene and overexpressed it using standard techniques (4, 17). S. aureus RN4220 coaA was amplified by PCR, introducing an NdeI site at the start codon and an XhoI site after the stop codon, and cloned into pSTBlue1 using the Perfectly Blunt Cloning kit. The gene was excised by digestion with NdeI and XhoI and ligated into similarly digested pET28a. The final construct encoded the N-terminal six-His-tagged S. aureus CoaA.Tuner (DE3) cells were transformed with this construct and grown at 37°C in Luria-Bertani medium-50-g/ml kanamycin. Protein expression was induced by 500 M isopropylthio-␤-Dgalactoside, and cells were harvested 3 h postinduction. The cell pellet was resuspended and sonicated, and cell debris was removed by centrifugation. The supernatant was subjected to Ni-chelating column chromatography followed by a HiTrap Q Sepharose ion exchange column. Enzyme identity was confirmed through N-terminal sequencing and matrix-assisted...

Feedback Inhibition of Pantothenate Kinase Regulates Pantothenol Uptake by the Malaria Parasite

“…Inhibitors of the conversion of pantothenate to CoA are being investigated both as antimalarial (3,(15)(16)(17) and antibacterial (12)(13)(14) agents. Pantothenol, a pantothenate analog and a common ingredient of multivitamin supplements, shampoos, and cosmetics, is one such candidate that has been shown recently to inhibit P. falciparum growth in vitro and to inhibit the in vivo proliferation of the mouse parasite Plasmodium vinckei vinckei.…”

“…NMR spectra were recorded on a Bruker Avance 800 NMR spectrometer operating at 800. 13 MHz. The chemical shifts (␦) are reported as the shift in ppm from trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (0.00 ppm).…”

“…Recent recognition of the sequence dissimilarity between prokaryotic and eukaryotic CoA biosynthesis enzymes (11) has renewed interest in the CoA biosynthesis pathway as a target for the development of novel antibacterial agents (12)(13)(14). Similarly, recent evidence indicates that malaria parasite proteins required for pantothenate transport and metabolism hold promise as muchneeded chemotherapeutic targets (15).…”

Acyl Carrier Protein Is a Cellular Target for the Antibacterial Action of the Pantothenamide Class of Pantothenate Antimetabolites

“…Whereas the coaA gene characterized in E. coli is widespread, Staphylococcus aureus possesses a pantothenate kinase whose primary sequence is distinct from its E. coli counterpart (12.7% identical). Nonetheless, this version of pantothenate kinase is also inhibited by N5-Pan and N7-Pan, and the analogs are toxic to S. aureus (31). Other bacteria such as Pseudomonas aeruginosa and Helicobacter pylori do not have a recognizable pantothenate kinase gene in their chromosomes, although they clearly have the other four genes that comprise the CoA biosynthetic pathway (32,33).…”

Prokaryotic type III pantothenate kinase enhances coenzyme A biosynthesis in <i>Escherichia coli</i>

“…The eukaryotic enzyme called PanK comprises a fourth category (Choudhry et al, 2003;Leonardi et al, 2005b). The eukaryotic and prokaryotic type I enzymes are highly regulated through end-product inhibition by CoA and its derivatives (Calder et al, 1999;Vallari et al, 1987).…”