Elmendorf Laura D, Hall Ryan L, Costa Flavia G, Escalante-Semerena Jorge C, Brunold Thomas C
Department of Chemistry, University of Wisconsin─Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States.
Department of Microbiology, University of Georgia─Athens, Cedar Street Building C, Athens, Georgia 30602, United States.
J Am Chem Soc. 2025 Jan 22;147(3):2380-2392. doi: 10.1021/jacs.4c11488. Epub 2025 Jan 8.
The adenosylcobalamin (AdoCbl)-dependent enzyme ethanolamine ammonia-lyase (EAL) catalyzes the conversion of ethanolamine to acetaldehyde and ammonia. As is the case for all AdoCbl-dependent isomerases, the catalytic cycle of EAL is initiated by homolytic cleavage of the cofactor's Co-C bond, producing Cocobalamin (CoCbl) and an adenosyl radical that serves to abstract a hydrogen atom from the substrate. Remarkably, in the presence of substrate, the rate of Co-C bond homolysis of enzyme-bound AdoCbl is increased by 12 orders of magnitude. For Class I AdoCbl-dependent isomerases, an important contribution to this rate acceleration stems from a stabilization of the CoCbl posthomolysis product by the axially coordinated histidine residue that displaces the pendant base from the Co ion upon AdoCbl binding to these enzymes. However, EAL and other Class II isomerases bind AdoCbl in the so-called "base-on" conformation and must therefore employ a different mechanism of Co-C bond activation. In the present study, we have used a combined spectroscopic and computational approach to probe the conformational changes and enzyme/cofactor/substrate interactions that contribute to the rate acceleration of Co-C bond homolysis in EAL. Spectroscopic data of AdoCbl and CoCbl show minimal perturbations upon cofactor binding to EAL in both the absence and presence of substrate. Structural models of free and EAL-bound AdoCbl were constructed using molecular dynamics and quantum mechanics/molecular mechanics computations. By carrying out relaxed potential energy scans for Co-C bond cleavage of free and EAL-bound AdoCbl, we identified key cofactor/enzyme interactions that contribute to the Co-C bond activation by EAL and obtained Co-C bond dissociation energies that agree well with published experimental data.
腺苷钴胺素（AdoCbl）依赖性酶乙醇胺氨裂解酶（EAL）催化乙醇胺转化为乙醛和氨。与所有AdoCbl依赖性异构酶的情况一样，EAL的催化循环由辅因子的Co-C键均裂引发，产生钴胺素（CoCbl）和一个腺苷自由基，该自由基用于从底物中提取一个氢原子。值得注意的是，在底物存在的情况下，酶结合的AdoCbl的Co-C键均裂速率增加了12个数量级。对于I类AdoCbl依赖性异构酶，这种速率加速的一个重要贡献源于轴向配位的组氨酸残基对CoCbl均裂后产物的稳定作用，当AdoCbl与这些酶结合时，该组氨酸残基会将悬垂碱基从Co离子上取代。然而，EAL和其他II类异构酶以所谓的“碱基朝上”构象结合AdoCbl，因此必须采用不同的Co-C键激活机制。在本研究中，我们使用了光谱学和计算相结合的方法来探究构象变化以及酶/辅因子/底物相互作用，这些因素有助于EAL中Co-C键均裂的速率加速。AdoCbl和CoCbl的光谱数据显示，在有无底物的情况下，辅因子与EAL结合时的扰动最小。使用分子动力学和量子力学/分子力学计算构建了游离和EAL结合的AdoCbl的结构模型。通过对游离和EAL结合的AdoCbl的Co-C键断裂进行松弛势能扫描，我们确定了有助于EAL激活Co-C键的关键辅因子/酶相互作用，并获得了与已发表的实验数据非常吻合的Co-C键解离能。
