Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, UK.
Nat Commun. 2024 Mar 4;15(1):1956. doi: 10.1038/s41467-024-46123-z.
Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical N-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BH1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BH1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BH1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.
定向进化已为计算设计酶提供了新的见解，揭示了蛋白质中复杂催化位点的出现机制。在这方面，我们最近表明，组氨酸亲核试剂和柔性精氨酸可以协同作用，以无与伦比的效率加速 Morita-Baylis-Hillman（MBH）反应。在这里，我们表明，用非经典的 N-甲基组氨酸（MeHis23）亲核试剂替代催化组氨酸会导致明显不同的进化结果，其中催化精氨酸 124 已被放弃。相反，出现了Glu26，它介导限速质子转移步骤，提供了一种酶（BH1.8），其活性比我们早期的 MBHase 高一个数量级以上。有趣的是，尽管 BH1.8 中的 MeHis23 到 His 的取代使活性降低了 4 倍，但产生的含 His 的变体仍然是一种有效的 MBH 生物催化剂。然而，对 BH1.8 进化轨迹的分析表明，MeHis 亲核试剂在工程早期解锁新的机制途径中至关重要。该研究表明，即使对设计酶的关键催化元件进行细微的扰动，也会导致截然不同的进化结果，从而为复杂化学反应提供新的机制解决方案。
