TY - JOUR
T1 - 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
AU - Tolentino Collado, Jinnette
AU - Bodis, Emoke
AU - Pasitka, Jonatan
AU - Szucs, Mihaly
AU - Fekete, Zsuzsanna
AU - Kis-Bicskei, Nikolett
AU - Telek, Elek
AU - Pozsonyi, Kinga
AU - Kapetanaki, Sofia M.
AU - Greetham, Greg
AU - Tonge, Peter J.
AU - Meech, Stephen R.
AU - Lukacs, Andras
N1 - 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.).
PY - 2024/3/1
Y1 - 2024/3/1
N2 - 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.
AB - 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.
U2 - 10.1016/j.jmb.2023.168312
DO - 10.1016/j.jmb.2023.168312
M3 - Article
SN - 0022-2836
VL - 436
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
M1 - 168312
ER -