Összes szerző

Meech Stephen R.

az alábbi absztraktok szerzői között szerepel:

Lukács András
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

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P17

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 

Emőke Bódis¹, Jinnette Tolentino Collado², Mihály Szűcs¹, Zsuzsanna Fekete¹, Elek Telek¹, Kinga Pozsonyi¹, Sofia M. Kapetanaki¹, Greg Greetham³, Peter J. Tonge², Stephen R. Meech⁴, and András Lukács¹

¹ Department of Biophysics, Medical School, University of Pécs, 7624 Pécs, Hungary

² Department of Chemistry, Stony Brook University, New York, 11794, United States.

³ Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, U.K

 ⁴ School of Chemistry, University of East Anglia, Norwich, NR4 7TJ, U.K.

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 [1-3]. The light-induced changes in the BLUF domain are transduced via a mechanism that has not been established yet to the adenylate cyclase domain. One critical residue in the vicinity of the flavin in BLUF proteins is the glutamine amino acid close to the N5 of the flavin[4-6]. 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 have applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation decelerated the primary electron transfer process but switched the enzyme in to a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the wild-type OaPAC. Differential scanning calorimetry measurements pointed out to a less compact structure for the mutant. These findings provide insight into the important elements in PACs and how their fine tuning may result to the design of optogenetic devices.

Acknowledgment

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.

References

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[2] Ohki M, Sugiyama K, Kawai F, Tanaka H, Nihei Y, Unzai S, et al. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium. Proceedings of the National Academy of Sciences of the United States of America. 2016;113:6659-64.

[3] Collado J, Iuliano J, Pirisi K, Jewlikar S, Adamczyk K, Greetham G, et al. Unraveling the Photoactivation Mechanism of a Light-Activated Adenylyl Cyclase Using Ultrafast Spectroscopy Coupled with Unnatural Amino Acid Mutagenesis. Acs Chemical Biology. 2022;17:2643-54.

[4] Udvarhelyi A, Domratcheva T. Glutamine rotamers in BLUF photoreceptors: a mechanistic reappraisal. J Phys Chem B. 2013;117:2888-97.

[5] Domratcheva T, Hartmann E, Schlichting I, Kottke T. Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry. Sci Rep. 2016;6:22669.

[6] Hontani Y, Mehlhorn J, Domratcheva T, Beck S, Kloz M, Hegemann P, et al. Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction. J Am Chem Soc. 2023;145:1040-52.