Elucidation and Molecular Characterization of the N-terminal Adenylyl Cyclase Activity of a Recombinant Nucleotide Binding Site Protein from Arabidopsis thaliana

Abstract

Adenylate cyclases (ACs) are a diverse group of enzymes, which catalyze the formation of 3',5' cyclic adenosine monophosphate (cAMP) from adenosine 5'-triphosphate (ATP). Cyclic AMP, just like the other known cyclic mononucleotides, is a key secondary messenger that is known to be involved in the mediation of responses to various extracellular stimuli in nearly all living organisms. Recently, a total of 14 probable candidate AC proteins in the Arabidopsis thaliana genome were identified and proposed through a bioinformatics approach based on the functionally assigned amino acid residues in the catalytic centre of annotated nucleotide cyclases. Among these identified candidates, was the putative truncated nucleotide binding site (NBS) protein (22 kDa), which harbours a novel AC catalytic centre, and is encoded by the At3g14460 gene. To date and incidentally, there are only six annotated and experimentally confirmed ACs in higher plants, which are the Zea mays pollen protein, the A. thaliana pentatricopeptide repeat protein, the Nicotiana benthamiana adenylyl cyclase protein, the Hippeastrum hybridum adenylyl cyclase protein, the A. thaliana K+-uptake permease 7 protein and the A. thaliana clathrin assembly protein. Therefore, and with a view to attempt and identify yet another additional higher plant AC molecule, this study was set out to clone, partially express and functionally characterize the annotated NBS protein in form of its truncated version harbouring the annotated AC catalytic center (the NBS-AC protein). This was achieved by germinating A. thaliana seeds, extracting total RNA from the A. thaliana plants, and isolating and transcribing the NBS-AC gene fragment. The transcribed gene fragment was resolved on agarose gel through electrophoresis. The amplified NBS-AC gene fragment was ligated into a pTrcHis2-TOPO® expression vector to optimize the cloning techniques. A complementation test was then performed to determine the ability of the NBSAC to complement the AC levels of the cyaA SP850 mutant in Escherichia coli cells. Following the complementation test, various bioinformatics tools were used to determine the level of expression of NBS in Arabidopsis. Findings from both the chemical and bioinformatics approaches, unequivocally demonstrated that this putative protein candidate is indeed a bona fide functional higher plant AC with a role in cAMP-regulated pathogen response and disease resistance.