Mechanism of photocontrol via switchable unnatural amino acids in asparaginase-glutaminases

• Applicant: Dr. Andrea Hupfeld
• Subject Area: Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 539618613

The reversible spatio-temporal control of enzymes with light by incorporation of photoswitchable unnatural amino acids (UAAs) is of growing interest for various biological, biotechnological and industrial applications. However, the engineering of these enzymes, dubbed switchable “photoxenases”, is limited by the lack of knowledge on the underlying molecular mechanism of photocontrol. Within this project, a dual approach will be implemented to investigate the principles of photocontrol in photoxenases. To this end, the asparaginase-glutaminase EcAII from Escherichia coli will be used as it meets all three criteria we set on a model system: i) It exhibits an inactive and an active conformation, which appears to ease the engineering of switchable photoxenases; ii) It follows an induced-fit catalytic mechanism supported by available comprehensive biochemical and structural data; and iii) Photocontrol of its glutaminase activity is of potential significance for its application as chemotherapeutic agent. The project will be structured into a “bottom up” and a “top down” approach to analyze the relationship between the light-regulation factor (LRF) as a measure of photocontrol efficiency and distinct enzymatic reaction steps. In particular, we intend to clarify whether the photoswitch affects only one catalytic reaction step or multiple catalytic reaction steps such as the substrate-induced loop closure or the chemical step. In the bottom up approach, we plan to use stopped-flow pre-steady state kinetic measurements as well as diverse biochemical analyses to identify the reaction steps, which are affected by photocontrol in two previously established EcAII-photoxenases. The resolution of this approach is restricted by the moderate LRF of 3–7 and the heterogeneity in UAA configuration of the two EcAII-photoxenases. Hence, we will employ a second, complementary top down approach, in which we will alter the catalytic reaction steps by two protein engineering strategies and analyze the resulting changes in the LRF. While investigative directed evolution will allow us to introduce targeted modifications in specific enzymatic reaction steps, the replacement of the EcAII scaffold with homologous asparaginase-glutaminases will establish more drastic alterations of the catalytic reaction. Ultimately, we will unite the findings of the two approaches in a combined evaluation to draft a holistic model for the photocontrol of asparaginase-glutaminases. By this, we hope to contribute novel insights that might improve the engineering of switchable photoxenases.

• DFG Programme: Research Grants