Kinateder Thomas, Drexler Lukas, Duran Cristina, Osuna Sílvia, Sterner Reinhard
Institute of Biophysics and Physical Biochemistry, Regensburg Center for Biochemistry, University of Regensburg, Regensburg, Germany.
Institut de Química Computacional i Catàlisi (IQCC) and Departament de Química, Universitat de Girona, Girona, Spain.
Protein Sci. 2025 Apr;34(4):e70103. doi: 10.1002/pro.70103.
Allosteric regulation of catalytic activity is a widespread property of multi-enzyme complexes. The tryptophan synthase is a prototypical allosteric enzyme where the constituting α (TrpA) and β (TrpB) subunits mutually activate each other in a manner that is incompletely understood. Experimental and computational studies have shown that LBCA-TrpB from the last bacterial common ancestor contains six residues (Res) distal from the active site that allow for high stand-alone catalytic activity in the absence of a TrpA subunit. In the present study, a database search revealed that Res is also present in the extant plTrpB from Pelodictyon luteolum. The plTrpB enzyme showed a high stand-alone activity and only a moderate activation by plTrpA. The replacement of LBCA-Res in plTrpB with the consensus residues from a multiple sequence alignment yielded plTrpB-con, which showed a dramatically decreased stand-alone activity but was strongly stimulated by plTrpA. These findings suggest that the effect of these six key allosteric residues is largely independent of the protein context within a specific TrpB enzyme. Analysis of the conformational landscapes of plTrpB and plTrpB-con revealed that plTrpB in isolation displays efficient closure of both the active site and the communication (COMM) domain. In contrast, these catalytically competent states are destabilized in plTrpB-con but can be recovered by the addition of plTrpA. A correlation-based shortest path map (SPM) analysis reveals that the catalytically and allosterically relevant domains-specifically, the COMM domain in TrpB and loops 2 and 6 in TrpA-are tightly interconnected exclusively in plTrpA:plTrpB-con.
催化活性的变构调节是多酶复合物广泛具有的特性。色氨酸合酶是一种典型的变构酶，其组成的α（TrpA）和β（TrpB）亚基以一种尚未完全理解的方式相互激活。实验和计算研究表明，来自最后一个细菌共同祖先的LBCA-TrpB在活性位点远端含有六个残基（Res），在没有TrpA亚基的情况下，这些残基可实现较高的独立催化活性。在本研究中，数据库搜索显示，来自黄绿颤藻的现存plTrpB中也存在Res。plTrpB酶表现出较高的独立活性，且仅被plTrpA适度激活。用多序列比对得到的共有残基替换plTrpB中的LBCA-Res，得到plTrpB-con，其独立活性显著降低，但受到plTrpA的强烈刺激。这些发现表明，这六个关键变构残基的作用在很大程度上独立于特定TrpB酶内的蛋白质环境。对plTrpB和plTrpB-con构象景观的分析表明，孤立的plTrpB能有效地封闭活性位点和通讯（COMM）结构域。相比之下，这些具有催化活性的状态在plTrpB-con中不稳定，但可通过添加plTrpA恢复。基于相关性的最短路径图（SPM）分析表明，催化和变构相关结构域——具体而言，TrpB中的COMM结构域以及TrpA中的环2和环6——仅在plTrpA:plTrpB-con中紧密相连。
