title: Allantoinase and dihydroorotase binding and inhibition by flavonols and the substrates of cyclic amidohydrolases.
abstract: Allantoinase and dihydroorotase are members of the cyclic amidohydrolases family. Allantoinase and dihydroorotase possess very similar binuclear metal centers in the active site and may use a similar mechanism for catalysis. However, whether the substrate specificities of allantoinase and dihydroorotase overlap and whether the substrates of other cyclic amidohydrolases inhibit allantoinase and dihydroorotase remain unknown. In this study, the binding and inhibition of allantoinase (Salmonella enterica serovar Typhimurium LT2) and dihydroorotase (Klebsiella pneumoniae) by flavonols and the substrates of other cyclic amidohydrolases were investigated. Hydantoin and phthalimide, substrates of hydantoinase and imidase, were not hydrolyzed by allantoinase and dihydroorotase. Hydantoin and dihydroorotate competitively inhibited allantoinase, whereas hydantoin and allantoin bind to dihydroorotase, but do not affect its activity. We further investigated the effects of the flavonols myricetin, quercetin, kaempferol, and galangin, on the inhibition of allantoinase and dihydroorotase. Allantoinase and dihydroorotase were both significantly inhibited by kaempferol, with IC50 values of 35 ± 3 μM and 31 ± 2 μM, respectively. Myricetin strongly inhibited dihydroorotase, with an IC50 of 40 ± 1 μM. The double reciprocal of the Lineweaver-Burk plot indicated that kaempferol was a competitive inhibitor for allantoinase but an uncompetitive inhibitor for dihydroorotase. A structural study using PatchDock showed that kaempferol was docked in the active site pocket of allantoinase but outside the active site pocket of dihydroorotase. These results constituted a first study that naturally occurring product flavonols inhibit the cyclic amidohydrolases, allantoinase, and dihydroorotase, even more than the substrate analogs (>3 orders of magnitude). Thus, flavonols may serve as drug leads for designing compounds that target several cyclic amidohydrolases.
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan; School of Medicine, College of Medicine, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan.
authors.forename: Wei-Feng
authors.initials: WF
authors.lastname: Peng
authors.affiliation: School of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan; Department of Medical Research, Chung Shan Medical University Hospital, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung City, Taiwan. Electronic address: cyhuang@csmu.edu.tw.
authors.forename: Cheng-Yang
authors.initials: CY
authors.lastname: Huang
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001667:Binding, Competitive; D020134:Catalytic Domain; D004080:Dihydroorotase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006827:Hydantoins / Q000737:chemistry; D044949:Kaempferols / Q000737:chemistry*; D007711:Klebsiella pneumoniae / Q000201:enzymology; D008958:Models, Molecular; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D010797:Phthalimides / Q000737:chemistry; D011485:Protein Binding; D012486:Salmonella typhimurium / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006827:Hydantoins; D044949:Kaempferols; D010797:Phthalimides; C004768:4,5-dihydroorotic acid; C037431:phthalimide; D009963:Orotic Acid; C006552:kaempferol; D000581:Amidohydrolases; D004080:Dihydroorotase; C019659:allantoinase
keywords: Allantoinase; Amidohydrolase; Dihydroorotase; Hydantoinase; Imidase