Photocycle alteration and increased enzymatic activity in genetically modified photoactivated adenylate cyclase OaPAC

Katalin Raics, Katalin Pirisi, Bo Zhuang, Zsuzsanna Fekete, Nikolett Kis-Bicskei, Ildiko Pecsi, Kinga Pozsonyi Ujfalusi, Elek Telek, Yin Li, Jinnette Tolentino Collado, Peter J. Tonge, Stephen R. Meech, Marten H. Vos, Emoke Bodis, Andras Lukacs

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

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.

Original languageEnglish
Article number105056
JournalThe Journal of Biological Chemistry
Volume299
Issue number8
Early online date17 Jul 2023
DOIs
Publication statusPublished - Aug 2023

Keywords

  • BLUF domain
  • cAMP
  • flavin
  • PCET
  • photoactivated adenylate cyclase
  • photochemistry
  • proton-coupled electron transfer
  • transient absorption
  • transient fluorescence

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