Gilead Sciences and IOCB Research Centre, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic; Department of Biochemistry, Faculty of Sciences, Charles University in Prague, Czech Republic.
FEBS J. 2014 Jul;281(14):3228-42. doi: 10.1111/febs.12857. Epub 2014 Jun 17.
In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.
除了在中枢神经系统中具有特征明显的作用外，人类谷氨酸羧肽酶 II（GCPII；Uniprot ID Q04609）在小肠中作为叶酸水解酶发挥作用，参与膳食多聚谷氨酸化叶酸（叶酸 - n-γ-L-谷氨酸）的吸收，这是叶酸的维生素前体形式（也称为维生素 B9）。尽管 GCPII 作为叶酸水解酶发挥作用，但在结构或酶学水平上，尚未了解 GCPII 对多聚谷氨酸化叶酸的加工。此外，许多关于天然存在的 His475Tyr 多态性与叶酸状态关系的流行病学研究表明，这种多态性可能与几种与叶酸代谢受损相关的病理有关。在本研究中，我们报告：（a）一系列与一组天然多聚谷氨酸化叶酸结合的催化失活 GCPII 突变体（Glu424Ala）复合物的 X 射线结构；（b）分辨率为 1.83 Å 的 His475Tyr 变体的 X 射线结构；（c）通过突变（Arg463Leu、Arg511Leu 和 Trp541Ala）、抑制剂结合和酶动力学研究最近发现的 GCPII 芳基结合位点，使用多聚谷氨酸化叶酸作为底物；（d）GCPII 野生型和 His475Tyr 变体的热稳定性和叶酸水解活性的比较。结果，晶体学数据揭示了多聚谷氨酸化叶酸与 GCPII 结合模式的大量细节，特别是芳基结合位点在识别叶酸部分中的参与。此外，综合结构和动力学数据表明，GCPII 野生型和 His475Tyr 变体在功能上是相同的。
