Kneuttinger, Andrea C. and Zwisele, Stefanie and Straub, Kristina and Bruckmann, Astrid and Busch, Florian and Kinateder, Thomas and Gaim, Barbara and Wysocki, Vicki H. and Merkl, Rainer and Sterner, Reinhard (2019) Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 20 (20): 5106. ISSN , 1422-0067
Full text not available from this repository. (Request a copy)Abstract
The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of similar to 100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | 3-DIMENSIONAL STRUCTURE; OPTICAL CONTROL; AMINO-ACID; WILD-TYPE; ACTIVATION; INDOLE; COMMUNICATION; MECHANISM; COMPLEX; BIOCATALYSIS; allostery; biocatalysis; enzymology; photo-control; protein design; unnatural amino acids |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie I > Prof. Dr. Gunter Meister |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 30 Mar 2020 09:19 |
| Last Modified: | 30 Mar 2020 09:19 |
| URI: | https://pred.uni-regensburg.de/id/eprint/26029 |
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