Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2011 Jul 1;286(26):23357-67. doi: 10.1074/jbc.M111.221069. Epub 2011 May 3.
The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.
10-甲酰四氢叶酸脱氢酶（FDH，ALDH1L1）的 C 端结构域（C(t)-FDH）是一种 NADP(+)依赖型氧化还原酶，也是醛脱氢酶的结构和功能同源物。在这里，我们报告了几种 C(t)-FDH 突变体的晶体结构，其中与结合的 NADP(+)烟酰胺环相邻的两个必需催化残基，半胱氨酸 707 和谷氨酸 673，分别或同时被替换。用丙氨酸替换谷氨酸会导致辅酶不可逆结合，而烟酰胺环附近没有明显的构象变化。进一步用丙氨酸替换半胱氨酸 707（E673A/C707A 双突变体）不会影响这种不可逆结合，表明缺乏谷氨酸是导致酶与辅酶之间增强相互作用的唯一原因。用丙氨酸替换半胱氨酸不影响 NADP(+)的结合，但导致酶失去区分氧化和还原辅酶的能力：与野生型 C(t)-FDH/NADPH 复合物不同，在 C707A 突变体中，NADPH 的位置与 NADP(+)的位置相同，烟酰胺环在催化中心内排列整齐。因此，谷氨酸限制了对辅酶的亲和力，而半胱氨酸是辅酶氧化还原状态的传感器。这些结论通过辅酶结合实验得到了证实。我们的研究进一步表明，辅酶的结合还受到催化中心和辅酶结合域之间长程通讯的控制，并指出参与这种通讯的一个α-螺旋涉及腺嘌呤部分的结合。
