For the plus-enzyme control, an UMP assay was performed in the absence of inhibitor. In the minus-enzyme control, sterile water instead of enzyme was used. The IC50s were calculated using a linear regression standard curve to predict the concentration of compound needed for 50% inhibition. One unit of activity was defined as the amount of enzyme required to degrade 0.1 nmol of ATP in learn more 120 min at 30 °C under the conditions described above. The minimum inhibitory concentrations (MICs) were determined by a standard microdilution broth method (National Committee for Clinical Laboratory Standards, 2003) with slight modifications. Briefly, the inoculum

size was ~5 × 105 CFU mL−1 in the final assay volume of 50 μL. The microdilution plates inoculated with bacteria were incubated at 35 °C for 18–20 h, and

the MIC was determined as the lowest concentration of the compound that completely inhibited the viable growth of the organism in the microdilution wells. Equilibrium analysis by SPR was performed using a Biacore3000 and the CM5 sensor chip (GE Healthcare Anti-infection Compound Library price Japan). SpPyrH was covalently coupled to CM5 using a standard amine coupling method according to the manufacturer’s protocol. Briefly, CM5 was activated by injecting a mixture of 20 mM N-hydroxysuccinimide (NHS) and 80 mM 1-ethyl-3- (3-diethylaminopropyl) carbodiimide hydrochloride. After being diluted tenfold with acetate buffer (pH 4.8), SpPyrH (0.1 mg mL−1) was injected at 10 μL min−1 for 7 min and then CM5 was inactivated by 1 M ethanolamine hydrochloride

(pH 8.5) to block the residual NHS ester groups. Running buffer (10 mM Hepes (pH7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Surfactant (GE Healthcare Japan), 5% DMSO) was used in all binding experiments. All compounds dissolved in DMSO were diluted 1 : 20 with the running buffer without 5% DMSO. The samples were injected at 30 μL min−1 Fossariinae for 2 min. The response was measured in resonance units (RU), and data analysis of the sensorgrams was performed using BIAevaluation software ver. 3.1 and the response at the equilibrium phase of interaction was obtained using the software program ‘equilibrium analysis model’. To obtain recombinant PyrH proteins, the SpPyrH or HiPyrH, each tagged with 6xHis at NH2-terminus, was expressed in E. coli and then purified using the Ni-affinity resin. When purified SpPyrH or HiPyrH protein was examined by SDS–PAGE followed by Coomassie staining, a prominent band was detected of 29.2 or 28.3 kDa in size, respectively, which was deduced as the molecular weight of SpPyrH or HiPyrH (Fig. 1a and c). These proteins were also detected by Western blotting analysis with anti-6xHis antibody, suggesting that each of these proteins is an authentic target protein (Fig. 1b and d).