Wieland, Mona and Luizaga, Jonnely and Duran, Cristina and Germscheid, Barbara and Rein, Johanna and Bruckmann, Astrid and Hiefinger, Caroline and Osuna, Silvia and Hupfeld, Andrea (2025) Reversible Substrate-Specific Photocontrol of the Chemotherapeutic Asparaginase(-Glutaminase) from Escherichia coli. ACS CATALYSIS, 15 (10). pp. 8462-8478. ISSN 2155-5435,
Full text not available from this repository. (Request a copy)Abstract
Photoswitchable unnatural amino acids are valuable engineering tools in biotechnology, particularly for the reversible control of enzymes with light. Here, we explore some basic principles of this protein engineering technique to simplify its approach and increase its success rate. To this end, we have selected Escherichia coli type II asparaginase (EcAII), which is a prominent chemotherapeutic enzyme that is limited by detrimental side effects associated with its promiscuous glutaminase activity. Incorporation of phenylalanine-4 '-azobenzene (AzoF) combined with extensive biophysical characterizations identified two light-sensitive variants, in which glutamine hydrolysis could be reversibly (de)activated up to 9-fold, whereas asparaginase hydrolysis was only marginally light-responsive. Computationally determined conformational landscapes elucidated this substrate-specificity of photocontrol defining a clear engineering principle: An exchange between less and more productive states at the active site helps AzoF to reshape the conformational landscape and makes enzymes more susceptible toward photocontrol. Moreover, our findings mark EcAII-AzoF variants as potential chemotherapeutic precursors.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ACTIVE-SITE; AMINO-ACID; CRYSTAL-STRUCTURE; L-ASPARAGINASE; ENZYME; PROTEINS; DYNAMICS; SOFTWARE; CHARGES; <sc>l</sc>-asparaginase; photocontrol; photoswitches; protein engineering; unnatural amino acids; molecular dynamics simulations; conformational landscapes |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 02 Jun 2026 09:30 |
| Last Modified: | 02 Jun 2026 09:30 |
| URI: | https://pred.uni-regensburg.de/id/eprint/65910 |
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