Chemical screening with a malachite green assay method

The screening of about 150 chemical compounds (Table S1) was performed with a malachite green assay method¹⁸. The principle of this method is: when SNTs transfer the AMP moiety from ATP to the heptose, ADP-d-glycero-β-d-manno-heptose and pyrophosphate (PPi) are produced. PPi was decayed into two phosphates by inorganic pyrophosphatase (IPP) and phosphates were measured by the malachite green method. β-d-Glucose-1-phosphate (βG1P) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan); TCI was used as a substrate because it is difficult to obtain the actual substrate, d-glycero-β-d-manno-heptose-1-phosphate (βH1P). The content of the α-form of this product was less than 0.1% in the current lot. A colour reagent of the malachite green method for phosphate detection was a mixture of ammonium molybdate ((NH₄)₆Mo₇O₂₄), malachite green solution and Tween 20 in the ratio 1:3:0.1. The mixture was filtered with a PVDF syringe filter and stood at room temperature (RT) for 1 h before use. All chemicals (25 μM) were tested for their inhibitory potential through a comparison of actual absorbances with control at 620 nm. The actual absorbance was obtained from the difference in absorbance between the reaction mixtures with and without BpHldC. The reaction mixture included 10 mM Tris–HCl (pH 7.5), 10 mM MgCl₂, 0.04 unit IPP, and 0.025 mg ml⁻¹ BpHldC (1.3 µM). To evaluate the accuracy of the inhibitor screening, Z′ factor was determined to be 0.9 (n = 15). The results indicate that the accuracy of the enzyme inhibitor test using this method is excellent¹⁹.