Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter January 9, 2019

Characterization and engineering of photoactivated adenylyl cyclases

  • Birthe Stüven , Robert Stabel , Robert Ohlendorf , Julian Beck , Roman Schubert and Andreas Möglich ORCID logo EMAIL logo
From the journal Biological Chemistry


Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3′, 5′-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.


We thank members of the Möglich laboratory for discussions, Dr. Manuela Stierl for providing the pBADM-30-bPAC plasmid, Dr. Tilo Mathes for advice on generating the CmpX13 ΔcyaA knockout strain, Norbert Grillenbeck for technical support, and Dr. Markus Lippitz for advice on the design of the LED matrix. We are indebted to the electrical workshop at the University of Bayreuth for circuit-board design and overall stellar support. Funding through Boehringer-Ingelheim Fonds (R.O.), funder id: 10.13039/100005156, a Sofja-Kovalevskaya Award (to A.M.) by the Alexander-von-Humboldt Foundation, and Deutsche Forschungsgemeinschaft (funder id: 10.13039/501100001659, grant MO21921/4-1) is gratefully acknowledged.


Andersen, K.R., Leksa, N.C., and Schwartz, T.U. (2013). Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins Struct. Funct. Bioinform. 81, 1857–1861.10.1002/prot.24364Search in Google Scholar PubMed PubMed Central

Andrée, B., Hillemann, T., Kessler-Icekson, G., Schmitt-John, T., Jockusch, H., Arnold, H.-H., and Brand, T. (2000). Isolation and characterization of the novel Popeye gene family expressed in skeletal muscle and heart. Dev. Biol. 223, 371–382.10.1006/dbio.2000.9751Search in Google Scholar PubMed

Ashman, D.F., Lipton, R., Melicow, M.M., and Price, T.D. (1963). Isolation of adenosine 3′, 5′-monophosphate and guanosine 3′, 5′-monophosphate from rat urine. Biochem. Biophys. Res. Commun. 11, 330–334.10.1016/0006-291X(63)90566-7Search in Google Scholar

Avelar, G.M., Schumacher, R.I., Zaini, P.A., Leonard, G., Richards, T.A., and Gomes, S.L. (2014). A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus. Curr. Biol. 24, 1234–1240.10.1016/j.cub.2014.04.009Search in Google Scholar PubMed PubMed Central

Blain-Hartung, M., Rockwell, N.C., Moreno, M.V., Martin, S.S., Gan, F., Bryant, D.A., and Lagarias, J.C. (2018). Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells. J. Biol. Chem. 293, 8473–8483.10.1074/jbc.RA118.002258Search in Google Scholar PubMed PubMed Central

Datsenko, K.A. and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640–6645.10.1073/pnas.120163297Search in Google Scholar PubMed PubMed Central

de Rooij, J., Zwartkruis, F.J.T., Verheijen, M.H.G., Cool, R.H., Nijman, S.M.B., Wittinghofer, A., and Bos, J.L. (1998). Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature 396, 474–477.10.1038/24884Search in Google Scholar PubMed

Deisseroth, K., Feng, G., Majewska, A.K., Miesenböck, G., Ting, A., and Schnitzer, M.J. (2006). Next-generation optical technologies for illuminating genetically targeted brain circuits. J. Neurosci. 26, 10380–10386.10.1523/JNEUROSCI.3863-06.2006Search in Google Scholar PubMed PubMed Central

Escobar, F.V., Buhrke, D., Michael, N., Sauthof, L., Wilkening, S., Tavraz, N.N., Salewski, J., Frankenberg-Dinkel, N., Mroginski, M.A., Scheerer, P., et al. (2017). Common structural elements in the chromophore binding pocket of the Pfr state of bathy phytochromes. Photochem. Photobiol. 93, 724–732.10.1111/php.12742Search in Google Scholar PubMed

Etzl, S., Lindner, R., Nelson, M.D., and Winkler, A. (2018). Structure-guided design and functional characterization of an artificial red light–regulated guanylate/adenylate cyclase for optogenetic applications. J. Biol. Chem. 293, 9078–9089.10.1074/jbc.RA118.003069Search in Google Scholar PubMed PubMed Central

Fesenko, E.E., Kolesnikov, S.S., and Lyubarsky, A.L. (1985). Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 313, 310–313.10.1038/313310a0Search in Google Scholar PubMed

Gao, S., Nagpal, J., Schneider, M.W., Kozjak-Pavlovic, V., Nagel, G., and Gottschalk, A. (2015). Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp. Nat. Commun. 6, 8046.10.1038/ncomms9046Search in Google Scholar PubMed PubMed Central

Gasser, C., Taiber, S., Yeh, C.-M., Wittig, C.H., Hegemann, P., Ryu, S., Wunder, F., and Möglich, A. (2014). Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. Proc. Natl. Acad. Sci. USA 111, 8803–8808.10.1073/pnas.1321600111Search in Google Scholar PubMed PubMed Central

Gibson, D.G., Young, L., Chuang, R.-Y., Venter, J.C., Hutchison, C.A., and Smith, H.O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345.10.1038/nmeth.1318Search in Google Scholar PubMed

Gleichmann, T., Diensthuber, R.P., and Möglich, A. (2013). Charting the signal trajectory in a light-oxygen-voltage photoreceptor by random mutagenesis and covariance analysis. J. Biol. Chem. 288, 29345–29355.10.1074/jbc.M113.506139Search in Google Scholar PubMed PubMed Central

Gold, M.G., Gonen, T., and Scott, J.D. (2013). Local cAMP signaling in disease at a glance. J. Cell Sci. 126, 4537–4543.10.1242/jcs.133751Search in Google Scholar PubMed PubMed Central

Gomelsky, M. (2011). cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use them all! Mol. Microbiol. 79, 562–565.10.1111/j.1365-2958.2010.07514.xSearch in Google Scholar PubMed PubMed Central

Gomelsky, M. and Klug, G. (2002). BLUF: a novel FAD-binding domain involved in sensory transduction in microorganisms. Trends Biochem. Sci. 27, 497–500.10.1016/S0968-0004(02)02181-3Search in Google Scholar

Görke, B. and Stülke, J. (2008). Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nat. Rev. Microbiol. 6, 613–624.10.1038/nrmicro1932Search in Google Scholar PubMed

Hennemann, J., Iwasaki, R.S., Grund, T.N., Diensthuber, R.P., Richter, F., and Möglich, A. (2018). Optogenetic control by pulsed illumination. ChemBioChem 19, 1296–1304.10.1002/cbic.201800030Search in Google Scholar PubMed

Iseki, M., Matsunaga, S., Murakami, A., Ohno, K., Shiga, K., Yoshida, K., Sugai, M., Takahashi, T., Hori, T., and Watanabe, M. (2002). A blue-light-activated adenylyl cyclase mediates photoavoidance in Euglena gracilis. Nature 415, 1047–1051.10.1038/4151047aSearch in Google Scholar PubMed

Jansen, V., Alvarez, L., Balbach, M., Strünker, T., Hegemann, P., Kaupp, U.B., and Wachten, D. (2015). Controlling fertilization and cAMP signaling in sperm by optogenetics. eLife 4, e05161.10.7554/eLife.05161Search in Google Scholar PubMed PubMed Central

Jansen, V., Jikeli, J.F., and Wachten, D. (2017). How to control cyclic nucleotide signaling by light. Curr. Opin. Biotechnol. 48, 15–20.10.1016/j.copbio.2017.02.014Search in Google Scholar PubMed

Jenal, U., Reinders, A., and Lori, C. (2017). Cyclic di-GMP: second messenger extraordinaire. Nat. Rev. Microbiol. 15, 271–284.10.1038/nrmicro.2016.190Search in Google Scholar PubMed

Kim, T., Folcher, M., Baba, M.D.-E., and Fussenegger, M. (2015). A synthetic erectile optogenetic stimulator enabling blue-light-inducible penile erection. Angew. Chem. Int. Ed. 54, 5933–5938.10.1002/anie.201412204Search in Google Scholar PubMed

Krauss, G. (2014). Biochemistry of Signal Transduction and Regulation (Weinheim, Germany: Wiley-VCH).10.1002/9783527667475Search in Google Scholar

Kuo, J.F. and Greengard, P. (1970). Cyclic nucleotide-dependent protein kinases. VI. Isolation and partial purification of a protein kinase activated by guanosine 3′,5′-monophosphate. J. Biol. Chem. 245, 2493–2498.10.1016/S0021-9258(18)63097-2Search in Google Scholar

Losi, A., Gardner, K.H., and Möglich, A. (2018). Blue-light receptors for optogenetics. Chem. Rev. 118, 10659–10709.10.1021/acs.chemrev.8b00163Search in Google Scholar PubMed PubMed Central

Malan, T.P. and McClure, W.R. (1984). Dual promoter control of the Escherichia coli lactose operon. Cell 39, 173–180.10.1016/0092-8674(84)90203-4Search in Google Scholar PubMed

Marden, J.N., Dong, Q., Roychowdhury, S., Berleman, J.E., and Bauer, C.E. (2011). Cyclic GMP controls Rhodospirillum centenum cyst development. Mol. Microbiol. 79, 600–615.10.1111/j.1365-2958.2010.07513.xSearch in Google Scholar PubMed PubMed Central

Mathes, T., Vogl, C., Stolz, J., and Hegemann, P. (2009). In vivo generation of flavoproteins with modified cofactors. J. Mol. Biol. 385, 1511–1518.10.1016/j.jmb.2008.11.001Search in Google Scholar PubMed

McDonough, K.A. and Rodriguez, A. (2012). The myriad roles of cyclic AMP in microbial pathogens: from signal to sword. Nat. Rev. Microbiol. 10, 27–38.10.1038/nrmicro2688Search in Google Scholar PubMed PubMed Central

Möglich, A. (2018). An open-source, cross-platform resource for nonlinear least-squares curve fitting. J. Chem. Educ. 95, 2273–2278.10.1021/acs.jchemed.8b00649Search in Google Scholar

Möglich, A., Ayers, R.A., and Moffat, K. (2009). Design and signaling mechanism of light-regulated histidine kinases. J. Mol. Biol. 385, 1433–1444.10.1016/j.jmb.2008.12.017Search in Google Scholar PubMed PubMed Central

Möglich, A., Yang, X., Ayers, R.A., and Moffat, K. (2010). Structure and function of plant photoreceptors. Annu. Rev. Plant Biol. 61, 21–47.10.1146/annurev-arplant-042809-112259Search in Google Scholar PubMed

Mukougawa, K., Kanamoto, H., Kobayashi, T., Yokota, A., and Kohchi,T. (2006). Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli. FEBS Lett. 580, 1333–1338.10.1016/j.febslet.2006.01.051Search in Google Scholar PubMed

Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K., and Möglich, A. (2012). From dusk till dawn: one-plasmid systems for light-regulated gene expression. J. Mol. Biol. 416, 534–542.10.1016/j.jmb.2012.01.001Search in Google Scholar PubMed

Ohlendorf, R., Schumacher, C.H., Richter, F., and Möglich, A. (2016). Library-aided probing of linker determinants in hybrid photoreceptors. ACS Synth. Biol. 5, 1117–1126.10.1021/acssynbio.6b00028Search in Google Scholar PubMed

Raffelberg, S., Wang, L., Gao, S., Losi, A., Gärtner, W., and Nagel, G. (2013). A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420. Biochem. J. 455, 359–365.10.1042/BJ20130637Search in Google Scholar PubMed

Rall, T.W., Sutherland, E.W., and Berthet, J. (1957). The relationship of epinephrine and glucagon to liver phosphorylase Iv. Effect of epinephrine and glucagon on the reactivation of phosphorylase in liver homogenates. J. Biol. Chem. 224, 463–475.10.1016/S0021-9258(18)65045-8Search in Google Scholar

Rauch, A., Leipelt, M., Russwurm, M., and Steegborn, C. (2008). Crystal structure of the guanylyl cyclase Cya2. Proc. Natl. Acad. Sci. USA 105, 15720–15725.10.1073/pnas.0808473105Search in Google Scholar PubMed PubMed Central

Richter, F., Scheib, U.S., Mehlhorn, J., Schubert, R., Wietek, J., Gernetzki, O., Hegemann, P., Mathes, T., and Möglich, A. (2015). Upgrading a microplate reader for photobiology and all-optical experiments. Photochem. Photobiol. Sci. 14, 270–279.10.1039/C4PP00361FSearch in Google Scholar PubMed

Richter, F., Fonfara, I., Bouazza, B., Schumacher, C.H., Bratovič, M., Charpentier, E., and Möglich, A. (2016). Engineering of temperature- and light-switchable Cas9 variants. Nucleic Acids Res. 44, 10003–10014.10.1093/nar/gkw930Search in Google Scholar PubMed PubMed Central

Rink, T.J., Tsien, R.Y., and Pozzan, T. (1982). Cytoplasmic pH and free Mg2+ in lymphocytes. J. Cell Biol. 95, 189–196.10.1083/jcb.95.1.189Search in Google Scholar PubMed PubMed Central

Rockwell, N.C. and Lagarias, J.C. (2010). A brief history of phytochromes. Chemphyschem Eur. J. Chem. Phys. Phys. Chem. 11, 1172–1180.10.1002/cphc.200900894Search in Google Scholar PubMed PubMed Central

Roychowdhury, S., Dong, Q., and Bauer, C.E. (2015). DNA-binding properties of a cGMP-binding CRP homologue that controls development of metabolically dormant cysts of Rhodospirillum centenum. Microbiology 161, 2256–2264.10.1099/mic.0.000172Search in Google Scholar PubMed PubMed Central

Ryu, M.-H. and Gomelsky, M. (2014). Near-infrared light responsive synthetic c-di-GMP module for optogenetic applications. ACS Synth. Biol. 3, 802–810.10.1021/sb400182xSearch in Google Scholar PubMed PubMed Central

Ryu, M.-H., Moskvin, O.V., Siltberg-Liberles, J., and Gomelsky, M. (2010). Natural and engineered photoactivated nucleotidyl cyclases for optogenetic applications. J. Biol. Chem. 285, 41501–41508.10.1074/jbc.M110.177600Search in Google Scholar PubMed PubMed Central

Ryu, M.-H., Kang, I.-H., Nelson, M.D., Jensen, T.M., Lyuksyutova, A.I., Siltberg-Liberles, J., Raizen, D.M., and Gomelsky, M. (2014). Engineering adenylate cyclases regulated by near-infrared window light. Proc. Natl. Acad. Sci. USA 111, 10167–10172.10.1073/pnas.1324301111Search in Google Scholar PubMed PubMed Central

Ryu, M.-H., Youn, H., Kang, I.-H., and Gomelsky, M. (2015). Identification of bacterial guanylate cyclases. Proteins 83, 799–804.10.1002/prot.24769Search in Google Scholar PubMed PubMed Central

Scheib, U., Stehfest, K., Gee, C.E., Körschen, H.G., Fudim, R., Oertner, T.G., and Hegemann, P. (2015). The rhodopsin–guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling. Sci. Signal 8, rs8.10.1126/scisignal.aab0611Search in Google Scholar PubMed

Schröder-Lang, S., Schwärzel, M., Seifert, R., Strünker, T., Kateriya, S., Looser, J., Watanabe, M., Kaupp, U.B., Hegemann, P., and Nagel, G. (2007). Fast manipulation of cellular cAMP level by light in vivo. Nat. Methods 4, 39–42.10.1038/nmeth975Search in Google Scholar PubMed

Schumacher, C.H., Körschen, H.G., Nicol, C., Gasser, C., Seifert, R., Schwärzel, M., and Möglich, A. (2016). A fluorometric activity assay for light-regulated cyclic-nucleotide-monophosphate actuators. Methods Mol. Biol. 1408, 93–105.10.1007/978-1-4939-3512-3_7Search in Google Scholar PubMed

Shimada, T., Fujita, N., Yamamoto, K., and Ishihama, A. (2011). Novel Roles of cAMP Receptor Protein (CRP) in Regulation of Transport and Metabolism of Carbon Sources. PLoS One 6, e20081.10.1371/journal.pone.0020081Search in Google Scholar PubMed PubMed Central

Shu, X., Royant, A., Lin, M.Z., Aguilera, T.A., Lev-Ram, V., Steinbach, P.A., and Tsien, R.Y. (2009). Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807.10.1126/science.1168683Search in Google Scholar PubMed PubMed Central

Stierl, M., Stumpf, P., Udwari, D., Gueta, R., Hagedorn, R., Losi, A., Gärtner, W., Petereit, L., Efetova, M., Schwarzel, M., et al. (2011). Light-modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium beggiatoa. J. Biol. Chem. 286, 1181–1188.10.1074/jbc.M110.185496Search in Google Scholar PubMed PubMed Central

Strack, R.L., Strongin, D.E., Bhattacharyya, D., Tao, W., Berman, A., Broxmeyer, H.E., Keenan, R.J., and Glick, B.S. (2008). A noncytotoxic DsRed variant for whole-cell labeling. Nat. Methods 5, 955–957.10.1117/12.808046Search in Google Scholar

Walsh, D.A., Perkins, J.P., and Krebs, E.G. (1968). An adenosine 3′,5′-monophosphate-dependant protein kinase from rabbit skeletal muscle. J. Biol. Chem. 243, 3763–3765.10.1016/S0021-9258(19)34204-8Search in Google Scholar

Yang, X., Kuk, J., and Moffat, K. (2008). Crystal structure of Pseudomonas aeruginosa bacteriophytochrome: Photoconversion and signal transduction. Proc. Natl. Acad. Sci. USA 105, 14715–14720.10.1073/pnas.0806718105Search in Google Scholar PubMed PubMed Central

Ziegler, T. and Möglich, A. (2015). Photoreceptor engineering. Front. Mol. Biosci. 2, 30.10.3389/fmolb.2015.00030Search in Google Scholar PubMed PubMed Central

Received: 2018-09-15
Accepted: 2018-12-07
Published Online: 2019-01-09
Published in Print: 2019-02-25

©2019 Walter de Gruyter GmbH, Berlin/Boston

Downloaded on 28.2.2024 from
Scroll to top button