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.

Jinnette Tolentino Collado, Emoke Bodis, Jonatan Pasitka, Mihaly Szucs, Zsuzsanna Fekete, Nikolett Kis-Bicskei, Elek Telek, Kinga Pozsonyi, Sofia M Kapetanaki, Greg Greetham, Peter J Tonge, Stephen R Meech, Andras Lukacs

Journal: Journal of molecular biology 2024;436(5):168312

PMID: 37827329

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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.

Address: Department of Chemistry, Stony Brook University, New York 11794, United States.; Department of Biophysics, Medical School, University of Pecs, Szigeti str. 12, 7624 Pecs, Hungary.; Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot OX11 0QX, UK.; Department of Chemistry, Stony Brook University, New York 11794, United States. Electronic address: [email protected].; School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, UK. Electronic address: [email protected].; Department of Biophysics, Medical School, University of Pecs, Szigeti str. 12, 7624 Pecs, Hungary. Electronic address: [email protected].

DOI: 10.1016/j.jmb.2023.168312

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MeSH Terms: Adenylyl Cyclases, Bacterial Proteins, Flavins, Light, Mutation, Glutamine, Protein Domains, Electron Transport, Enzyme Activation, Oscillatoria