Fig 1.
Substrate specificity of dihydropyrimidinase.
(A) Substrate of dihydropyrimidinase, hydantoinase, imidase, allantoinase, and dihydroorotase. Kinetic analysis of dihydropyrimidinase was carried out using (B) dihydrouracil, (C) phthalimide, and (D) 5-propyl-hydantoin as a substrate. The maximal concentration of phthalimide was limited to 1 mM due to its poor solubility. Data points are an average of 2–3 determinations within 10% error.
Table 1.
Primers used for construction of plasmids.
Table 2.
Effect of metal ions on the activity of dihydropyrimidinase.
Table 3.
Apparent Michaelis–Menten constants for dihydropyrimidinase using the substrate of each enzyme in the cyclic amidohydrolase family.
Fig 2.
Effect of the substrate and the inhibitor of allantoinase or dihydroorotase on the activity of dihydropyrimidinase using (A) 5-propyl-hydantoin or (B) dihydrouracil as a substrate.
Acetohydroxamate and 3-amino-1,2,4-triazole solutions, whose pH values, were pre-adjusted to pH 8. When using dihydrouracil as a substrate, some compounds at high concentrations were difficult to determine the inhibitory effect on the activity of dihydropyrimidinase using the spectrophotometric assay at 230 nm.
Fig 3.
Inhibition of dihydropyrimidinase by flavonoids.
(A) Molecular structure of myricetin, dihydromyricetin, and myricitrin. (B) IC50 determination of flavonoids for dihydropyrimidinase using dihydrouracil as a substrate. (C) IC50 determination of flavonoids for dihydropyrimidinase using 5-propyl-hydantoin as a substrate. IC50 value for dihydropyrimidinase was directly determined using graphic analysis.
Fig 4.
The active site of dihydropyrimidinase.
(A) The active site of P. aeruginosa dihydropyrimidinase. According to the crystal structure of Saccharomyces kluyveri (PDB entry: 2FVK), residues H59, H61, K150, H183, H239, and D316 of P. aeruginosa dihydropyrimidinase shown in yellow were crucial for the assembly of the binuclear metal center within the active site; meanwhile, residues Y155, S289, and N337 shown in limon were crucial for substrate binding. The model was directly constructed by superimposing the modeled structure of P. aeruginosa dihydropyrimidinase with the crystal structure of S. kluyveri dihydropyrimidinase-dihydrouracil complex. Dihydrouracil generated from the complex is shown in light magenta. (B) An alignment consensus of 497 sequenced dihydropyrimidinase homologs by ConSurf reveals the degree of variability at each position along the primary sequence. Note that the positions involved in the assembly of the binuclear metal center within the active site and the substrate binding of P. aeruginosa dihydropyrimidinase are well conserved.
Fig 5.
Representation of the docking models of the dihydropyrimidinase complex from PatchDock.
(A) The binding mode of dihydropyrimidinase to dihydromyricetin. Dihydromyricetin interacted with I95 (light pink), S289 (limon), and D316 (yellow) of dihydropyrimidinase, in which S289 and D316 were found to be crucial for the catalytic activity of dihydropyrimidinase. (B) The binding mode of dihydropyrimidinase to myricetin. Myricetin interacted with N157 (light pink), Q194 (light pink), R212 (light pink), and N337 (limon), in which N337 was found to be crucial for the catalytic activity of dihydropyrimidinase.
Fig 6.
Dihydromyricetin is a competitive inhibitor for dihydropyrimidinase.
Kinetic study of dihydropyrimidinase with (open circles) and without dihydromyricetin (close circles). Inhibition of dihydropyrimidinase by dihydromyricetin (40 μM) resulted in Lineweaver–Burk plots where the lines are cross the y-axis at the similar point, indicating that dihydromyricetin is a competitive inhibitor for dihydropyrimidinase. Data points are an average of 2–3 determinations within 10% error.
Fig 7.
The fluorescence emission spectra of dihydropyrimidinase.
The decrease in intrinsic fluorescence of protein was measured at 334.5 nm upon excitation at 279 nm and 25°C with a spectrofluorimeter. (A) The fluorescence emission spectra of dihydropyrimidinase with dihydromyricetin of different concentrations (0–50 μM). The fluorescence intensity emission spectra of dihydropyrimidinase significantly quenched with dihydromyricetin. (B) The fluorescence emission spectra of dihydropyrimidinase with myricetin of different concentrations (0–50 μM). The fluorescence intensity emission spectra of dihydropyrimidinase significantly quenched with myricetin. (C) Fluorescence titrations of dihydromyricetin and myricetin with dihydropyrimidinase. An aliquot amount of dihydromyricetin and myricetin was individually added to the enzyme solution for each Kd. The Kd was obtained by the equation: ΔF = ΔFmax-Kd(ΔF/[compound]). Data points are an average of 2–3 determinations within 10% error.
Fig 8.
The fluorescence emission spectra of the N337A mutant.
(A) The fluorescence emission spectra of the N337A mutant with dihydromyricetin of different concentrations (0–50 μM). (B) The fluorescence emission spectra of the N337A mutant with myricetin of different concentrations (0–50 μM). (C) Fluorescence titrations of dihydromyricetin and myricetin with the N337A mutant. An aliquot amount of dihydromyricetin and myricetin was individually added to the enzyme solution for each Kd. The Kd was obtained by the equation: ΔF = ΔFmax-Kd(ΔF/[compound]). Data points are an average of 2–3 determinations within 10% error.