Recombinant Expression and Functional Testing of Candidate Adenylate Cyclase Domains

  • Oziniel Ruzvidzo
  • Bridget T. Dikobe
  • David T. Kawadza
  • Grace H. Mabadahanye
  • Patience Chatukuta
  • Lusisizwe Kwezi
Part of the Methods in Molecular Biology book series (MIMB, volume 1016)


Adenylate cyclases (ACs) are enzymes capable of converting adenosine-5′-triphosphate to cyclic 3′, 5′-­adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their product cAMP have firmly been established as important signalling molecules with important roles in several cellular signal transduction pathways. However, in higher plants, the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of generating cAMP. Recently a number of candidate AC-encoding genes in Arabidopsis thaliana have been proposed based on functionally assigned amino acids in the catalytic center of annotated and/or experimentally tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and recombinant expression of functional candidate AC domains using, as an example, the A. thaliana pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a complementation test, in vivo adenylate cyclase activity of candidate recombinant molecules can be prescreened and promising candidates can subsequently be further evaluated in an in vitro AC immunoassay.

Key words

Arabidopsis thaliana Pentatricopeptide (PPR) Adenylate cyclase (AC) Adenosine-5′-triphosphate (ATP) Cyclic 3′,5′-adenosine monophosphate (cAMP) Lactose fermenters Enzyme immunoassay 



This material is based upon work supported financially by the National Research Foundation but any opinion, findings and ­conclusions or recommendations expressed in this material are those of the author(s) and therefore the NRF does not accept any liability in regard thereto.


  1. 1.
    Robison GA, Butcher RW, Sutherland EW (1968) Cyclic AMP. Annu Rev Biochem 37:149–174PubMedCrossRefGoogle Scholar
  2. 2.
    Goodman DB, Rasmussen H, DiBella F et al (1970) Cyclic adenosine 3′:5′-monophosphate-stimulated phosphorylation of isolated neurotubule subunits. Proc Natl Acad Sci USA 67:652–659PubMedCrossRefGoogle Scholar
  3. 3.
    Gerisch G, Hülser D, Malchow D et al (1975) Cell communication by periodic cyclic-AMP pulses. Philos Trans R Soc Lond B Biol Sci 272:181–192PubMedCrossRefGoogle Scholar
  4. 4.
    Ashton AR, Polya GM (1978) Cyclic adenosine 3′:5′-monophosphate in axenic rye grass endosperm cell cultures. Plant Physiol 61:718–722PubMedCrossRefGoogle Scholar
  5. 5.
    Butcher RW, Baird CE, Sutherland EW (1968) Effects of lipolytic and antilipolytic substances on adenosine 3′,5′-monophosphate levels in ­isolated fat cells. J Biol Chem 243:1705–1712PubMedGoogle Scholar
  6. 6.
    Amrhein N (1977) The current status of cyclic AMP in higher plants. Annu Rev Plant Physiol 28:123–132CrossRefGoogle Scholar
  7. 7.
    Meier S, Ruzvidzo O, Morse M et al (2010) The Arabidopsis wall-associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes. PLoS One 5:e8904PubMedCrossRefGoogle Scholar
  8. 8.
    Kurosaki F, Nishi A (1993) Stimulation of ­calcium influx and calcium cascade by cyclic AMP in cultured carrot cells. Arch Biochem Biophys 302:144–151PubMedCrossRefGoogle Scholar
  9. 9.
    Carricarte VC, Bianchini GM, Muschietti JP et al (1988) Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa). Biochem J 249:807–811PubMedGoogle Scholar
  10. 10.
    Li W, Luan S, Schreiber SL et al (1994) Cyclic AMP stimulates K+ channel activity in mesophyll cells of Vicia faba L. Plant Physiol 106:957–961PubMedCrossRefGoogle Scholar
  11. 11.
    Moutinho A, Hussey PJ, Trewavas AJ et al (2001) cAMP acts as a second messenger in pollen tube growth and reorientation. Proc Natl Acad Sci USA 98:10481–10486PubMedCrossRefGoogle Scholar
  12. 12.
    DeYoung BJ, Innes RW (2006) Plant NBS-LRR proteins in pathogen sensing and host defense. Nat Immun 7:1243–1249CrossRefGoogle Scholar
  13. 13.
    Newton RP, Smith CJ (2004) Cyclic nucleotides. Phytochemistry 65:2423–2437PubMedCrossRefGoogle Scholar
  14. 14.
    Kwezi L, Meier S, Mungur L et al (2007) The Arabidopsis thaliana brassinosteroid receptor (AtBRI1) contains a domain that functions as a guanylyl cyclase in vitro. PLoS One 2:7CrossRefGoogle Scholar
  15. 15.
    Mulaudzi T, Ludidi N, Ruzvidzo O et al (2011) Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro. FEBS lett 585:2693–2697PubMedCrossRefGoogle Scholar
  16. 16.
    Gehring C (2010) Adenyl cyclases and cAMP in plant signaling—past and present. Cell Commun Signal 8:15PubMedCrossRefGoogle Scholar
  17. 17.
    Nakamura T, Schuster G, Sugiura G et al (2004) Chloroplast RNA-binding and pentatricopeptide repeat proteins. Biochem Soc Trans 32:571–574PubMedCrossRefGoogle Scholar
  18. 18.
    Kotera E, Tasaka M, Shikanai T (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433:326–330PubMedCrossRefGoogle Scholar
  19. 19.
    Schmitz-Linneweber C, Williams-Carrier R, Barkan A (2005) RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5′ region of mRNAs whose translation it activates. Plant Cell 17:2791–2804PubMedCrossRefGoogle Scholar
  20. 20.
    Kurien BT, Scofield RH (2009) Nonelec­trophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens to obtain 12 immunoblots. Methods Mol Biol 536:55–65PubMedCrossRefGoogle Scholar
  21. 21.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  22. 22.
    Tang WJ, Stanzel M, Gilman AG (1995) Truncation and alanine-scanning mutants of type I adenylyl cyclase. Biochemistry 34:14563–14572PubMedCrossRefGoogle Scholar
  23. 23.
    Tesmer JJ, Dessauer CW, Sunahara RK et al (2000) Molecular basis for P-site inhibition of adenylyl cyclase. Biochemistry 39:14464–14471PubMedCrossRefGoogle Scholar
  24. 24.
    Geng W, Wang Z, Zhang J et al (2005) Cloning and characterization of the human soluble adenylyl cyclase. Am J Physiol 288:C1305–C1316CrossRefGoogle Scholar
  25. 25.
    Oh M-H, Kim HS, Wu X et al (2012) Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling. Biochem J 443:515–523PubMedCrossRefGoogle Scholar
  26. 26.
    Chen Y, Cann MJ, Litvin TN et al (2000) Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor. Science 289:625–628PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Oziniel Ruzvidzo
    • 1
  • Bridget T. Dikobe
    • 1
  • David T. Kawadza
    • 1
  • Grace H. Mabadahanye
    • 1
  • Patience Chatukuta
    • 1
  • Lusisizwe Kwezi
    • 1
  1. 1.Department of Biological Sciences, School of Environmental and Health SciencesNorth-West UniversityMmabathoSouth Africa

Personalised recommendations