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has been cited by the following article:

• TITLE: A Putative Protein with No Known Function in Arabidopsis thaliana Harbors a Domain with Adenylyl Cyclase Activity

  AUTHORS: Katlego S. Sehlabane, Patience Chatukuta, Tshegofatso B. Dikobe, Enetia D. Bobo, Angela Sibanda, David T. Kawadza, Oziniel Ruzvidzo

  KEYWORDS: Arabidopsis thaliana, Adenylyl Cyclase, Enzyme Immunoassay, Mass Spectrometry, Computational Analysis

  JOURNAL NAME: American Journal of Plant Sciences, Vol.13 No.7, July 18, 2022

  ABSTRACT: Adenylyl cyclases (ACs) are a special group of enzymes that catalyze formation of the second messenger molecule, 3',5'-cyclic adenosine monophosphate (cAMP) from 5'-adenosine triphosphate (ATP). Apparently, even though cAMP is increasingly becoming an important signaling molecule in higher plants, the identification of plant ACs has somewhat remained slow. Here we report the recombinant cloning, partial expression and affinity purification of the truncated version (AtAC261-388) of a putative Arabidopsis thaliana protein (AtAC: At3g21465) followed by a demonstration of its inherent enzymatic activity as an AC. Currently, AtAC is not assigned any particular function in A. thaliana but simply annotated as an AC-like protein and, therefore, we targeted it for our study to establish if it is indeed a bona fide AC molecule. From our work, we firstly, show through enzyme immunoassaying and mass spectrometry that the recombinant AtAC261-388 can generate cAMP from ATP in vitro in a manganese-dependent manner that is activated by calcium and hydrogen carbonate. Secondly, we reveal through computational analysis that the AC center of AtAC is solvent-exposed, and amenable to the unhindered access of ATP as a substrate for catalysis. Lastly, we show that the recombinant AtAC261-388 can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli strain.