Thomas Midhun George, Jaber Sathik Rifayee Simahudeen Bathir, Christov Christo Z
Department of Chemistry, Michigan Technological University, Houghton, Michigan 49931, United States.
ACS Catal. 2024 Dec 5;14(24):18550-18569. doi: 10.1021/acscatal.4c04010. eCollection 2024 Dec 20.
The ethylene-forming enzyme (EFE) is a Fe(II)/2-oxoglutarate (2OG) and l-arginine (l-Arg)-dependent oxygenase that primarily decomposes 2OG into ethylene while also catalyzing l-Arg hydroxylation. While the hydroxylation mechanism in EFE is similar to other Fe(II)/2OG-dependent oxygenases, the formation of ethylene is unique. Various redesign strategies have aimed to increase ethylene production in EFE, but success has been limited, highlighting the need for alternate approaches. It is crucial to incorporate an accurate and comprehensive description of the integrative and multidimensional effects of the protein environment to enhance the redesign strategy in metalloenzymes, particularly in EFE. This involves understanding the role of the second coordination sphere (SCS) and long-range (LR) interacting residues, correlated motions, electronic structure, intrinsic electric field (IntEF), as well as the stabilization of transition states and reaction intermediates. In this study, we employ a molecular dynamics-based quantum mechanics/molecular mechanics approach to examine the integrative effects of the protein environment on reactions catalyzed by EFE variants from the first coordination sphere (FCS, D191E), SCS (A198V and R171A) and LR (E215A). The study uncovers how substitutions at different positions in EFE similarly impact the ethylene-forming reaction while posing distinct effects on the hydroxylation reaction. Results predict the effect of the variants in controlling the 2OG coordination mode in the Fe(II) center. Specifically, the study suggests that D191E uniquely prefers transitioning from an to an 2OG coordination mode before dioxygen binding. However, studies on the 2OG flip in the presence of approaching dioxygen and dioxygen binding in the D191E variant indicate that the 2OG flip might not be feasible in the 5C Fe(II) state. Calculations show the possibility of a hydrogen atom transfer (HAT)-assisted oxygen flip in EFE and its variants (other than D191E). MD simulations elucidate the characteristic dynamic change in the α7 region in the D191E variant that might contribute to its increased hydroxylation reaction. Results indicate the possibility of forming an ferryl from the IM2 (Fe(III)-partial bond intermediate) in the D191E variant. This alternative pathway from IM2 may also exist in WT EFE and other variants, which are yet to be explored. The study also delineates the impact of substitutions on the electronic structure and IntEF. Overall, the calculations support the idea that understanding the integrative and multidimensional effects of the protein environment on the reactions catalyzed by EFE variants provides the basics for improved enzyme redesign protocols of EFE to increase ethylene production. The results of this study will also contribute to the development of alternate redesign strategies for other metalloenzymes.
乙烯形成酶（EFE）是一种依赖于Fe（II）/2-氧代戊二酸（2OG）和L-精氨酸（L-Arg）的加氧酶，它主要将2OG分解为乙烯，同时也催化L-Arg羟基化。虽然EFE中的羟基化机制与其他依赖于Fe（II）/2OG的加氧酶相似，但乙烯的形成却是独特的。各种重新设计策略旨在提高EFE中的乙烯产量，但成效有限，这凸显了需要采用替代方法。至关重要的是，要纳入对蛋白质环境的综合和多维效应的准确而全面的描述，以增强金属酶，特别是EFE中的重新设计策略。这涉及了解第二配位层（SCS）和远程（LR）相互作用残基的作用、相关运动、电子结构、固有电场（IntEF），以及过渡态和反应中间体的稳定性。在本研究中，我们采用基于分子动力学的量子力学/分子力学方法，来研究蛋白质环境对来自第一配位层（FCS，D191E）、SCS（A198V和R171A）和LR（E215A）的EFE变体催化反应的综合效应。该研究揭示了EFE中不同位置的取代如何类似地影响乙烯形成反应，同时对羟基化反应产生不同的影响。结果预测了这些变体在控制Fe（II）中心的2OG配位模式方面的作用。具体而言，该研究表明，D191E在双氧结合之前独特地倾向于从一种2OG配位模式转变为另一种。然而，对D191E变体中接近双氧时2OG翻转以及双氧结合的研究表明，在5C Fe（II）状态下2OG翻转可能不可行。计算表明EFE及其变体（除D191E外）中存在氢原子转移（HAT）辅助的氧翻转可能性。分子动力学模拟阐明了D191E变体中α７区域的特征性动态变化，这可能有助于其增加的羟基化反应。结果表明在D191E变体中可能从IM2（Fe（III）-部分键中间体）形成一种铁氧中间体。从IM2的这种替代途径也可能存在于野生型EFE和其他变体中，有待进一步探索。该研究还描述了取代对电子结构和IntEF的影响。总体而言，这些计算支持这样一种观点，即了解蛋白质环境对EFE变体催化反应的综合和多维效应为改进EFE的酶重新设计方案以提高乙烯产量提供了基础。本研究结果也将有助于开发其他金属酶的替代重新设计策略。
