Spectrometric and in-silico assessment of the adenylyl cyclase activity of a recombinant clathrin assembly protein from Arabidopsis thaliana
Adenylate cyclases (ACs) are enzymes that catalyse formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Although cAMP is recognised as an important role player in signalling pathways in higher plants, ACs are still yet to be fully consolidated as there are currently only eight experimentally confirmed such molecules in higher plants. In this study and in an effort to provide additional information on what is currently available about ACs in higher plants, we report the recombinant expression of an Arabidopsis thaliana clathrin assembly protein (AtClAP; At1g68110) and characterization of its function by means of mass spectrometry and in silico analysis. This AtClAP protein was initially annotated bionformatically as a putative AC candidate, followed by its recent practical confirmation as an AC, However, its AC function has not yet been fully elucidated. Our findings from this study have established the AtClAP protein as part of several cellular response systems responsible for growth, development and response to various environmental stress challenges in plants. Our findings from this study have established the AtC1AP protein as part of several cellular response systems responsible for growth, development and response to various environmental stress challenges in plants. In our study we used the E. cloni BL21 (DE3) pLysS DUOs cells as the expression host strain of choice and as a result the desired and targeted recombinant AtClAP protein was successfully expressed at its expected molecular weight size of around 22.6 kDa. Consequently, our study succeeded to purify the expressed recombinant AtClAP protein using an established chromatographic approach. The successfully purified protein was assessed by mass spectrophotometry and this detected high cAMP activity and other molecular components. The detected molecular components are proved to be key elements of the cell signaling systems in most living organisms including plants- necessary for essential cellular processes such as growth, development and responses to various environmental stress factors. After determining the enzymatic capacity of the recombinant AtC1AP protein we wanted to analyse its functional role in plants by means of an in silico analysis. The findings indicated AtClAP protein to be co-expressed with a wide array of other proteins of which the 25 top most proteins were specifically involved in peroxisomal transport, photoperiodic flowering, lipid modification, to mention a few. These are proven key cellular processes essential for plant growth, development and responses to various environmental stress factors.