Abstract
Cyclic nucleotide, such as cyclic GMP, is a secondary messenger that regulates a wide range of biological process via the diverse signaling cascades. Photoactivated adenylyl cyclases (PACs), constituted of blue light utilizing flavin (BLUF) and cyclase homology domain (CHD), are used as an optogenetic tool to modulate the cyclic AMP (cAMP) level and to study cAMP-mediated signal transduction mechanisms. Here, we have engineered photoactivated adenylyl cyclases (PACs) from microbes to photoactivated guanylyl cyclases (PGCs) via mutagenesis of the substrate binding-specific residues in cyclase homology domain. We demonstrate purification, photodynamic, and detailed biochemical characterization of the engineered PGCs that can serve as optogenetic tool for manipulation of cGMP level in the cells. Engineered PGCs show typical BLUF photoreceptor properties with different recovery kinetics and varying light-regulated guanylyl cyclase activities.
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Acknowledgements
We thank the CIF staff, University of Delhi South Campus (UDSC), for sequencing and CD spectroscopy studies. Dr. Ravi Tandon, SBT, JNU is acknowledged for sharing lab resources.
Funding
We thank the SERB for research grant (SB/EMEQ-188/2013) and the Govt. of India and R&D, University of Delhi, for funding the project.
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SK conceived the research plan, interpreted the data, and wrote the manuscript. MT designed and performed all the experiments, analyzed the data, and wrote the manuscript. KS and PM performed reviewer’s suggested experiments.
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Highlights
• Engineered photoactivated guanylyl cyclases (PGCs) possess different photocycles with BLUF photoreceptor properties.
• Engineered photoactivated guanylyl cyclases (PGCs) show an increase in cGMP level upon irradiation with blue light.
• Engineered photoactivated guanylyl cyclases (PGCs) with varying amplitudes of guanylyl cyclase activity might be due to residues within the catalytic active site other than the three substrate-binding residues.
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Tanwar, M., Sharma, K., Moar, P. et al. Biochemical Characterization of the Engineered Soluble Photoactivated Guanylate Cyclases from Microbes Expands Optogenetic Tools. Appl Biochem Biotechnol 185, 1014–1028 (2018). https://doi.org/10.1007/s12010-018-2710-x
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DOI: https://doi.org/10.1007/s12010-018-2710-x