Bhagat, Ashok Kumar and Schlee, Sandra and Straub, Kristina and Nazet, Julian and Luckner, Patricia and Bruckmann, Astrid and Sterner, Reinhard (2022) Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase. ACS SYNTHETIC BIOLOGY, 11 (8). pp. 2846-2856. ISSN 2161-5063,
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The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-KSOONBY and stTrpE-Y4550NBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K500NBY and stTrpE-Y4SSONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | AMINO-ACID; PROTEIN; CHORISMATE; IDENTIFICATION; PHOTOCONTROL; MECHANISM; feedback inhibition; anthranilate synthase; genetic code expansion; photoresponsive noncanonical amino acid; tryptophan |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Reinhard Sterner |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 17 Oct 2023 14:53 |
| Last Modified: | 17 Oct 2023 14:53 |
| URI: | https://pred.uni-regensburg.de/id/eprint/56500 |
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