Elucidation and Functional Characterization of the C-terminal Adenylyl Cyclase Activity of a Recombinant LeucineRich Repeat Protein from Arabidopsis thaliana

Abstract

Adenylate cyclases (ACs) are cellular enzymes that catalyze the hydrolysis of adenosine 5'triphosphate (ATP) into 3',5'-cyclic adenosine monophosphate (cAMP), which has long been established to be a second messenger in cellular signalling pathways of bacteria, fungi, lower eukaryotes and animals. Second messengers such as the cAMP have a key role in linking environmental stimuli to physiological responses of various cells; although the presence of this cAMP and the enzymes that generate it has always been a topic of long disagreements and serious controversy in higher plants. In 2010, Gehring bioinformatically identified various plant molecules carrying the probable AC catalytic motif yet most of such molecules have not yet been practically and/or experimentally tested for their abilities to generate cAMP from ATP. One of such candidate molecules is the leucine-rich repeat (LRR) protein that is coded for by the At3g14460 gene. To prove that this candidate has adenylate cyclase activity, the total RNA was extracted from the 6-week old A. thaliana followed by amplification of the LRR gene fragment using RT-PCR with its sequence-specific primers. The coding sequence of the putative AC catalytic domain of this protein (LRR-AC) was cloned and then further expressed in E. coli. This was then followed by an in vivo activity evaluation through complementation test in which Escherichia coli cyaA SP850 mutant was used to determine the inherent AC activity of the LRR. Furthermore, various bioinformatics tools were used to elucidate and analyse the functionality of this protein in Arabidopsis and other related higher plants. Findings obtained demonstrated that the LRR-AC was a functional AC capable of catalysing the conversion of ATP into cAMP with an essential role cAMP-regulated abiotic and biotic stress response.