Recombinant Expression and Molecular Elucidation of the Dual Functional Properties of a Truncated Pentatricopeptide Repeat Protein from Arabidopsis thaliana
Dikobe, Bridget Tshegofatso
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Plants play essential roles in the general life systems of mankind even though they also tend to experience constant challenges during their own life cycles from periodic exposures to various environmental stimuli (e.g. light, hormones, pathogens, sugars, and wounding), which affect their productivity and developmental systems. While, mechanisms by which these plants use to detect and transduce such external signals into their internal cellular environments have not been elucidated. However, there is a need for them to be clearly understood so that they can then be manipulated for the ultimate benefit of mankind. Currently, a special group of plant molecules termed adenylate cyclases (ACs) have been the main focus. These are enzymes capable of catalyzing the conversion of adenosine 5'-triphosphate (ATP) to the second messenger, 3',5'-adenosine cyclic monophosphate (cAMP), which in turn is involved in a variety of physiological and developmental processes in a number of organisms. Despite the fact that the roles of both the AC and its product, cAMP have been extensively studied and documented in animals and lower eukaryotes, not much is known about ACs in plants even though the cAMP has been widely implicated in a number of cellular processes such as the cell cycle, responses to stressful environmental factors, defense responses, and activation of the protein kinases. To date, only five higher plant A Cs are known and these are the PSiP protein from Zea mays; the AtPPR-AC protein from Arabidopsis thaliana; the NbAC protein from Nicotiana benthamiana; the HpACl protein from Hippeastrum hybridum; and the AtKUP7 protein from Arabidopsis thaliana. Apparently, each of these five identified plant A Cs bear a single catalytic domain in the form of the characterized AC domain but a recent study has further identified, from Arabidopsis thaliana, a related protein molecule with two annotated catalytic domains; the AC and kinase domains. This protein molecule is termed a pentatricopeptide repeat protein (AtPPR) coded for by the Atlg62590 gene. Therefore, in an attempt to identify yet another additional functional AC in higher plants and also to elucidate the possible functionality of twin-domain proteins in plants, we targeted the AtPPR protein in this study. In this plan, we cloned and partially expressed its AC/kinase-containing domain fragment (AtPPR-AC/K) in competent E. cloni EXPRESS BL2 l (DE3) pLysS cells and demonstrated its ability to induce the generation of endogenous cAMP in these prokaryotic host cells. In addition, we also demonstrated a complementation of the mutant non-lactose fermenting cyaA SP850 E. coli cells by this recombinant protein to apparently ferment lactose, and as a result of this AtPPR-AC/K's ability to generate the most needed cAMP necessary for this process. Furthermore, we also managed to chemically purify this recombinant AtPPR-AC/K protein and determined its AC activity in vitro, and during which it was also firmly established that the recombinant AtPPR-AC/K was indeed a bona fide soluble AC (sAC), whose functional activities in plants are mediated by cAMP via a calmodulin-dependent signalling system. Lastly, the possible kinase activity of the recombinant AtPPR-AC/K was also assessed resulting in this protein being established as a bona fide functional kinase with the intrinsic trans-phosphorylation and auto-phosphorylation activities. In line with this, we thus managed to establish the AtPPR-AC/K as a bona fide bi-functional plant molecule, having both the AC and kinase activities. More so, this work also, undoubtedly, established that there is a cross-talking scenario between the two catalytic domains of the AtPPR-AC/K - an aspect that partly explains how this putative protein is functionally modulated in higher plants. Finally, this study also managed to establish the AtPPR protein as the sixth ever AC molecule to be identified and experimentally confirmed in higher plants, while at the same time, it becomes the first ever higher plant molecule to possess a dual catalytic function of being both an AC and a kinase.