Chu Wen-Ting, Nesbitt Natasha M, Gnatenko Dmitri V, Li Zongdong, Zhang Beibei, Seeliger Markus A, Browne Seamus, Mantle Timothy J, Bahou Wadie F, Wang Jin
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, P. R. China.
Department of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
Chemistry. 2017 Feb 3;23(8):1891-1900. doi: 10.1002/chem.201604517. Epub 2017 Jan 11.
Biliverdin reductase IXβ (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1) elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2) defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP complex; 3) provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.
胆绿素还原酶IXβ（BLVRB）是血红素代谢中的一种关键酶。最近在人类中的研究发现了一种功能丧失突变（Ser111Leu），该突变揭示了其在造血过程中的一个至关重要的作用。我们进行了实验和热力学建模研究，以进一步深入了解辅因子在调节BLVRB催化机制的底物可及性和蛋白质折叠特性方面的作用。通过分子动力学（MD）模拟进行的定点诱变确定了NAD(P)H依赖性构象变化通过形成“疏水口袋”对底物可及性的关键作用，同时鉴定了一个调节NADPH/NADH选择性的单一关键残基（Arg35）。Loop80和Loop120在脱辅基BLVRB（开放状态）中阻断疏水底物结合口袋，而辅因子结合后这些结构的移动导致“封闭”（催化活性）构象。涉及位于BLVRB活性位点核心的Ser111的突变会影响酶活性和热力学稳定性。这项工作1）通过活性位点氢键网络阐明了Ser111在酶催化和热力学稳定性中的关键作用；2）定义了一个超越二元BLVRB/NADP复合物晶体结构的脱辅基BLVRB动态模型；3）为受NAD(P)H调节的二元复合物的“相遇”和“平衡”状态提供了结构基础。
