Sugrue E, Scott C, Jackson C J
Research School of Chemistry, Australian National University, Canberra, Australia.
Commonwealth Scientific and Industrial Research Organisation, Canberra, Australia.
Org Biomol Chem. 2017 Jan 25;15(4):937-946. doi: 10.1039/c6ob02355j.
One of the central goals of protein design and engineering is to be able to accurately predict the effects of a mutation on stability and activity. However, the genetic context into which mutations are introduced can lead to complex interactions between the mutation and other amino acids and unpredictable, non-additive, effects. This phenomenon is known as intramolecular epistasis and has been shown to restrict evolutionary paths through laboratory directed evolution experiments and ancestral protein reconstruction, but has rarely been studied at a quantitative level in naturally evolving enzymes. Atrazine-specific and atrazine/ametryn bispecific triazine hydrolases (TrzN) have evolved in different bacterial strains over the past fifty years in response to the presence of the synthetic herbicides atrazine and ametryn. Here, we have investigated all 24 evolutionary trajectories that are possible from monofunctional to bispecific TrzN isoforms in terms of activity, stability, expression and structure. The results reveal that half of these trajectories are unviable due to inactive intermediates, with only 1/24 trajectories exhibiting consistent improvement in bispecificity. The most viable path requires the mutation of Gln241 to Glu241 first, which increases activity 3-fold with atrazine and 10-fold with ametryn, which is further optimised in subsequent evolutionary steps. The epistatic interactions between mutations, involving control of the pK of catalytic residues, the thermostability of the protein, and soluble expression are shown to be responsible for the bottlenecks in this evolutionary landscape. This comprehensive analysis of the evolution of bispecificity highlights the importance of epistasis in protein engineering and evolution, which makes identifying the correct sequence in which to combine mutations extremely important.
蛋白质设计与工程的核心目标之一是能够准确预测突变对稳定性和活性的影响。然而，引入突变的遗传背景可能导致突变与其他氨基酸之间产生复杂的相互作用，并产生不可预测的、非加性的效应。这种现象被称为分子内上位性，已通过实验室定向进化实验和祖先蛋白重建证明其会限制进化路径，但在自然进化的酶中很少在定量水平上进行研究。在过去五十年中，针对合成除草剂阿特拉津和莠灭净的存在，阿特拉津特异性和阿特拉津/莠灭净双特异性三嗪水解酶（TrzN）在不同细菌菌株中进化而来。在此，我们研究了从单功能到双特异性TrzN亚型的所有24种可能的进化轨迹，涉及活性、稳定性、表达和结构。结果表明，由于中间产物无活性，这些轨迹中有一半是不可行的，只有1/24的轨迹在双特异性方面表现出持续改善。最可行的路径是首先将Gln241突变为Glu241，这使阿特拉津的活性提高3倍，莠灭净的活性提高10倍，并在随后的进化步骤中进一步优化。突变之间的上位性相互作用，包括催化残基pK的控制、蛋白质的热稳定性和可溶性表达，被证明是造成这一进化格局中瓶颈的原因。对双特异性进化的全面分析突出了上位性在蛋白质工程和进化中的重要性，这使得确定组合突变的正确顺序极为重要。
