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Raics, Katalin, Pirisi, Katalin, Zhuang, Bo, Fekete, Zsuzsanna, Kis-Bicskei, Nikolett, Pecsi, Ildiko, Ujfalusi, Kinga Pozsonyi, Telek, Elek, Li, Yin, Collado, Jinnette Tolentino, Tonge, Peter J., Meech, Stephen R. 
ORCID: https://orcid.org/0000-0001-5561-2782, Vos, Marten H., Bodis, Emoke and Lukacs, Andras
(2023)
Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC.
The Journal of Biological Chemistry, 299 (8).
ISSN 0021-9258
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Abstract
Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated.
| Item Type: | Article |
|---|---|
| Additional Information: | Funding Information: J. T. C. was supported by the National Institutes of Health IMSD-MERGE (T32GM135746) and NY-CAPs IRACDA (K12-GM102778) Programs at Stony Brook University. B. Z. was supported by a PhD scholarship from the Chinese scholarship Council. A. L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021. This study was supported by the National Science Foundation (NSF) (MCB-1817837 to P. J. T.) and the EPSRC (EP/N033647/1 to S. R. M.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. |
| Uncontrolled Keywords: | bluf domain,camp,flavin,pcet,photoactivated adenylate cyclase,photochemistry,proton-coupled electron transfer,transient absorption,transient fluorescence,molecular biology,biochemistry,cell biology,2* ,/dk/atira/pure/subjectarea/asjc/1300/1312 |
| Faculty \ School: | Faculty of Science > School of Chemistry |
| UEA Research Groups: | Faculty of Science > Research Groups > Centre for Photonics and Quantum Science Faculty of Science > Research Groups > Chemistry of Light and Energy |
| Related URLs: | |
| Depositing User: | LivePure Connector |
| Date Deposited: | 24 Aug 2023 10:31 |
| Last Modified: | 30 Aug 2023 08:30 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/92921 |
| DOI: | 10.1016/j.jbc.2023.105056 |
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