Single amino acid mutation decouples photochemistry of the BLUF domain from the enzymatic function of OaPAC and drives the enzyme to a switched-on state

Tolentino Collado, Jinnette, Bodis, Emoke, Pasitka, Jonatan, Szucs, Mihaly, Fekete, Zsuzsanna, Kis-Bicskei, Nikolett, Telek, Elek, Pozsonyi, Kinga, Kapetanaki, Sofia M., Greetham, Greg, Tonge, Peter J., Meech, Stephen R. ORCID: https://orcid.org/0000-0001-5561-2782 and Lukacs, Andras (2024) Single amino acid mutation decouples photochemistry of the BLUF domain from the enzymatic function of OaPAC and drives the enzyme to a switched-on state. Journal of Molecular Biology, 436 (5). ISSN 0022-2836

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Abstract

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

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. A.L. acknowledges funding from the Hungarian National Research and Innovation Office (K-137557) and was supported by PTE ÁOK-KA-2021. E.T was supported by PTE ÁOK-KA-2022-09. This study was supported by the National Science Foundation (NSF) (MCB-1817837 to PJT) and the EPSRC (EP/N033647/1 to S.R.M.).
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024)
UEA Research Groups:	Faculty of Science > Research Groups > Centre for Photonics and Quantum Science Faculty of Science > Research Groups > Chemistry of Light and Energy
Depositing User:	LivePure Connector
Date Deposited:	17 Oct 2023 00:45
Last Modified:	25 Sep 2024 17:31
URI:	https://ueaeprints.uea.ac.uk/id/eprint/93323
DOI:	10.1016/j.jmb.2023.168312

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