Lauko Anna, Pellock Samuel J, Sumida Kiera H, Anishchenko Ivan, Juergens David, Ahern Woody, Jeung Jihun, Shida Alexander F, Hunt Andrew, Kalvet Indrek, Norn Christoffer, Humphreys Ian R, Jamieson Cooper, Krishna Rohith, Kipnis Yakov, Kang Alex, Brackenbrough Evans, Bera Asim K, Sankaran Banumathi, Houk K N, Baker David
Department of Biochemistry, University of Washington, Seattle, WA, USA.
Institute for Protein Design, University of Washington, Seattle, WA, USA.
Science. 2025 Apr 18;388(6744):eadu2454. doi: 10.1126/science.adu2454.
The design of enzymes with complex active sites that mediate multistep reactions remains an outstanding challenge. With serine hydrolases as a model system, we combined the generative capabilities of RFdiffusion with an ensemble generation method for assessing active site preorganization at each step in the reaction to design enzymes starting from minimal active site descriptions. Experimental characterization revealed catalytic efficiencies (/) up to 2.2 × 10 M s and crystal structures that closely match the design models (Cα root mean square deviations <1 angstrom). Selection for structural compatibility across the reaction coordinate enabled identification of new catalysts remove with five different folds distinct from those of natural serine hydrolases. Our de novo approach provides insight into the geometric basis of catalysis and a roadmap for designing enzymes that catalyze multistep transformations.
设计具有介导多步反应的复杂活性位点的酶仍然是一项极具挑战性的任务。以丝氨酸水解酶作为模型系统，我们将RFdiffusion的生成能力与一种用于评估反应各步骤中活性位点预组织的整体生成方法相结合，以便从最小的活性位点描述开始设计酶。实验表征显示催化效率（/）高达2.2×10 M s，且晶体结构与设计模型紧密匹配（Cα均方根偏差<1埃）。通过在反应坐标上选择结构兼容性，能够鉴定出具有五种不同于天然丝氨酸水解酶折叠方式的新型催化剂。我们的从头设计方法为催化的几何基础提供了见解，并为设计催化多步转化的酶提供了路线图。
