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...
A low volume (10 μL) kinetic fluorescent assay to evaluate human microsomal epoxide hydrolase (mEH) activity with the α‐cyano‐methyl ester substrate, PHOME (Analyt. Biochem. (2006) 355, 71–80), has been optimized. mEH was successfully cloned into a mammalian expression vector, expressed transiently in FreeStyle 293F cells, and purified by affinity chromatography (purity > 80%). In the presence of 5 μM PHOME, reaction rates were linearly proportional to added enzyme to 0.1 μM and the reactions were linear for up to 60 minutes with respect to product formation. The lowest discernable reaction rate of 5.5 nM/min was obtained with 10 nM mEH; with an assay signal:noise of 3:1. Optimized assay buffer contained 25 mM HEPES pH 7.0, 0.01% CHAPS, and 0.005% casein. Purified mEH activity was selectively inhibited with the addition of 50 mM ZnSO4 or with the known mEH inhibitor, Valpromide (IC50 = 1 mM and 5 μM, respectively), indicating that product formation was due solely to mEH. The potency of Valpromide was shown to give similar values in an alternative assay format using mEH enriched microsomes (IC50 = 8.0 μM). AUDA, a potent sEH inhibitor, was not an inhibitor of purified mEH. Thus, purified mEH provides a means to develop a low volume, high throughput assay to further our efforts towards developing inhibitors that can selectively discriminate sEH versus mEH activity.
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