Lee Eunjeong, McLeod Matthew J, Redzic Jasmina S, Marcolin Barbara, Thorne Robert E, Agarwal Pratul, Eisenmesser Elan Zohar
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO, United States.
Laboratory of Atomic and Solid-State Physics, Cornell University, Ithaca, NY, United States.
Front Mol Biosci. 2023 Aug 14;10:1244587. doi: 10.3389/fmolb.2023.1244587. eCollection 2023.
Biliverdin Reductase B (BLVRB) is an NADPH-dependent reductase that catalyzes the reduction of multiple substrates and is therefore considered a critical cellular redox regulator. In this study, we sought to address whether both structural and dynamics changes occur between different intermediates of the catalytic cycle and whether these were relegated to just the active site or the entirety of the enzyme. Through X-ray crystallography, we determined the apo BLVRB structure for the first time, revealing subtle global changes compared to the holo structure and identifying the loss of a critical hydrogen bond that "clamps" the R78-loop over the coenzyme. Amide and Cα chemical shift perturbations were used to identify environmental and secondary structural changes between intermediates, with more distant global changes observed upon coenzyme binding compared to substrate interactions. NMR relaxation rate measurements provided insights into the dynamic behavior of BLVRB during the catalytic cycle. Specifically, the inherently dynamic R78-loop that becomes ordered upon coenzyme binding persists through the catalytic cycle while similar regions experience dynamic exchange. However, the dynamic exchange processes were found to differ through the catalytic cycle with several groups of residues exhibiting similar dynamic responses. Finally, both local and distal structural and dynamic changes occur within BLVRB that are dependent solely on the oxidative state of the coenzyme. Thus, through a comprehensive analysis here, this study revealed structural and dynamic alterations in BLVRB through its catalytic cycle that are not simply relegated to the active site, but instead, are allosterically coupled throughout the enzyme.
胆红素还原酶B（BLVRB）是一种依赖NADPH的还原酶，可催化多种底物的还原反应，因此被认为是一种关键的细胞氧化还原调节因子。在本研究中，我们试图探讨在催化循环的不同中间体之间是否发生了结构和动力学变化，以及这些变化是否仅局限于活性位点或整个酶分子。通过X射线晶体学，我们首次确定了脱辅基BLVRB的结构，发现与全酶结构相比存在细微的整体变化，并确定了一个关键氢键的丢失，该氢键将R78环“固定”在辅酶上。利用酰胺和Cα化学位移扰动来识别中间体之间的环境和二级结构变化，与底物相互作用相比，辅酶结合时观察到更远距离的整体变化。NMR弛豫速率测量为BLVRB在催化循环中的动态行为提供了见解。具体而言，在辅酶结合时变得有序的固有动态R78环在催化循环中持续存在，而类似区域经历动态交换。然而，发现动态交换过程在催化循环中有所不同，几组残基表现出相似的动态响应。最后，BLVRB内发生的局部和远端结构及动态变化仅取决于辅酶的氧化状态。因此，通过本研究的全面分析，揭示了BLVRB在其催化循环中的结构和动态变化，这些变化并非仅局限于活性位点，而是在整个酶分子中通过变构相互偶联。
