Department of Beauty Science, National Taichung University of Science and Technology, Taichung City 403, Taiwan.
Department of Biomedical Sciences, Chung Shan Medical University, Taichung City 402, Taiwan.
Biomolecules. 2023 Jan 11;13(1):149. doi: 10.3390/biom13010149.
Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate -carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.
二氢乳清酸酶（DHOase）是嘧啶核苷酸生物合成途径中的第三酶。在哺乳动物中，DHOase 在三功能酶 CAD 中具有活性，CAD 还具有氨甲酰磷酸合成酶和天冬氨酸转氨甲酰酶的活性。在这项研究之前，尚不清楚 FDA 批准的临床药物 5-氟尿嘧啶（5-FU）是否可以与人类 CAD（huDHOase）的 DHOase 结构域结合。在这里，我们确定 huDHOase 是一种新的 5-FU 结合蛋白，从而将 5-FU 相互作用组扩展到该人类酶。为了研究 5-FU 与 huDHOase 结合的位置，我们解决了 1.97 Å 的复合物晶体结构（PDB ID 8GVZ）。为了进行比较，还确定了与苹果酸结合的 huDHOase 复合物的晶体结构（PDB ID 8GW0）。这两个非底物配体结合在 huDHOase 的活性部位。以前已经确定，底物 -carbamoyl-L-aspartate 要么结合在活性部位，要么从活性部位移开，但它是延伸到活性部位的环（环入模式）或从活性部位移开的环（环出模式）。DHOase 也通过环出模式结合非底物配体。与 DHOase 模型相反，我们的复合物结构表明，huDHOase 通过环入模式与 5-FU 或苹果酸结合。我们进一步使用定点突变和荧光猝灭法表征了 5-FU 与 huDHOase 的结合。考虑到环入模式，huDHOase 中的动态环应该是进一步设计抑制剂和临床化疗以抑制癌细胞系嘧啶生物合成的合适药物靶向位点。
