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.