Biochemical and Structural Characterization of the Adenylyl Cyclase Activity of a Fusion Epsin N-terminal Homology Protein from Arabidopsis thaliana

Abstract

Adenylate cyclases (ACs) are enzymes that are known to produce 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP) as a result of some associated extracellular stimulations. However, the question of whether or not cAMP does exist in plants has been an issue of debate for a while, mainly due to the less efficient methods employed to isolate this molecule and also because of its very low levels in plants. In contrast to plants, animals and lower eukaryotic ACs and their product cAMP have been firmly established as important signalling molecules with critical roles in cellular signal transduction pathways. Therefore, and in an effort to augment information currently known about ACs in higher plants, this study targeted an Epsin N-terminus homology protein from Arabidopsis thaliana (AtENTH), whose gene was initially annotated as a probable AC bioinformatically and then recently confirmed as a bona fide AC practically. The study recombinantly expressed the AtENTH protein followed by a comprehensive characterization of its enzymatic AC activity biochemically using the enzyme immunoassaying technique and structurally through structural modelling, simulations and molecular docking techniques. Findings from the study, technically confirmed the AtENTH protein as a multi-domain multi-functional higher plant AC, whose catalytic activity has a very strong specificity for ATP as its sole substrate compared to the other known organic triphosphates, and binding to it using the E residue located at position 2 within its catalytic AC center. The AtENTH ultimately catalyzes the conversion of this preferred ATP substrate into cAMP using the K residue located at position 14 of its catalytic center.