Time-resolved catalysis and allostery in tryptophan synthase as model for bi-enzyme machineries with an active sites-connecting product/substrate tunnel

• Applicants: Dr. Andrea Hupfeld; Professor Matthias Wilmanns, Ph.D.
• Subject Area: Biochemistry ; Structural Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 508072649

Allosteric bi-enzyme complexes with tunnel-mediated product/substrate transport between two active sites are intricate enzyme machineries that coordinate two catalytic steps in a highly sophisticated manner. Previous studies from extensive biochemical and structural biology approaches have obtained only limited insight into the mechanism of catalysis, the allosteric regulation of activity and the associated conformational transitions of these enzymes. Within this project, a novel strategy for the investigation of bi-enzyme complexes will be implemented, which is based on the combination of light-sensitive unnatural amino acids (UAA) and Time-Resolved serial crystal X-ray crystallography (TRX) for the timely resolution of catalysis, allostery and product/substrate transport. To this end, tryptophan synthase (TS) will be used as pilot system. The already available comprehensive biochemical and structural data on both catalytic reactions and allostery-based conformational transitions make this bi-enzyme complex exceptionally suited for our proof-of-concept studies. The project will be structured into three phases. In Phase I, the two research groups will acquire complementary experimental data. One group will engineer TS mutants using light-sensitive UAAs, which block conformational changes or the transport of product/substrates and release this blockage upon irradiation. These enzyme mutants will then be biochemically and biophysically characterized to select suitable candidates for further time-resolved structural investigations. The other group will meanwhile set up TRX experiments for TS by initiating the reaction through ligand diffusion. Finally, the engineered light-sensitive TS mutants will be structurally characterized with TRX by laser induced reaction initiation. Both TRX approaches will be complementary in terms of key experimental requirements such as homogeneity of initiation, precise control of the reaction starting point, reachable time intervals and overall feasibility of the experimental approach. In Phase II, both groups will interpret their data and combine their results into a complete time-resolved visualization or “movie” of TS catalysis and allostery. In Phase III, the findings will be analyzed to draw general conclusions regarding three topics of broad significance: i) the mechanism of other product/substrate active sites-connecting bi-enzyme complexes, ii) the design of smart, light-sensitive antibiotics towards TS, and iii) the evolution of TS catalysis and allostery.

• DFG Programme: Research Grants