5. Enzyme assay

HPPD activity was detected through the conversion of its product, homogentisate, to maleylacetoacetate, then catalyzed by HGD from Pseudomonas aeruginosa (PaHGD). The preparation of recombinant PaHGD protein was performed as previously mentioned.^5,6)

In this study, the assay for HPPD activity was carried out at a final volume of 1 mL in a semi-micro cuvette. The reaction mixture contained 980 µL of reaction solution (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.0), 2 mM L(+)-ascorbic acid, 10 µM FeSO₄, 50 nM HGD, 240 nM HPPD), and 20 µL of the substrate HPP. Reactions were initiated by adding the reaction solution to HPP in a semi-micro cuvette. The reactions were monitored at 320 nm using a UV-2600 spectrophotometer (Shimadzu, Kyoto, Japan) at 25°C for 5 min. To evaluate the inhibitory activity of the compound on HPPD, 10 µL of the compound was added to the reaction mixture before adding the mixture to HPP. For a dose-response study, inhibitors were added at final concentrations of 1, 10, 30, 70, and 1,000 nM in the assay with the AtHPPD enzyme, and those were added at final concentrations of 1, 10, 25, 50, 70, 100, and 1,000 nM in the assay with the OsHPPD enzyme. The reaction mixture without HPPD was used as a negative control. A reaction mixture without the compound was used as a positive control. Inhibition of HPPD activity was determined by comparison with the positive control.