A Study Of The Interaction Mechanism Of GTP With CodY Of Bacillus Anthracis | 68158
Journal of Medical Microbiology & Diagnosis
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Bacillus anthracis, a prioritized bioterrorism agent, is a Gram-positive, sporulating, non-motile, aerobic bacterium which
causes the fatal zoonotic disease, anthrax, with humans as contingent victims. CodY, a global transcriptional regulator,
controls diverse cellular activities such as metabolism, amino acid biosynthesis and transport systems, nitrogen uptake, motility,
sporulation, pellicle, and biofilm formation, and most importantly virulence in almost all low G+C gram-positive bacteria. In
B. anthracis, about 500 genes are perceived to be the targets of CodY, including the master regulator AtxA, which is pivotal to
the manifestation of toxic constituents; namely a lethal factor, edema factor and protective antigen. GTP and Branched Chain
Amino Acids are the metabolic effectors of CodY, which affects its DNA-binding ability. In order to gain an insight into the
interaction mechanism of CodY and GTP, of which scarce is known presently, we carried out an in vitro GTP binding assay.
We have demonstrated that CodY of B. anthracis binds to GTP. Homology modeling and sequence/structure analysis of CodY
of B. anthracis revealed conserved GTP binding residues. Interestingly, we found that the CodY of B. anthracis could undergo
autophosphorylation with GTP as a phosphoryl Group donor. Furthermore, the phosphorylation site mutant (Ser215 to Ala215)
of CodY failed to retain this autophosphorylation activity and hence is the critical residue involved in autophosphorylation. Since
the Ser215 lies in the Helix-turn-Helix DNA binding motif of CodY and is conserved amongst its homologs, autophosphorylation
may be speculated as a self-regulatory mechanism of CodY activity in the cell. Inquisitively, we proceeded to test the GTPase
activity of CodY by thin-layer chromatography and found that the recombinant protein could withal hydrolyze GTP, albeit
weakly, as quantified spectrophotometrically. Predicated on these findings, we conclude that in contrast to its homologs in other
organisms, CodY of B. anthracis exhibits unique biochemical attributes such as GTP hydrolysis and autophosphorylation, which
might be further exploited as a novel drug target.
Shikha Joon is a Ph D candidate with a particular interest in studying novel drug targets against infectious diseases. Prior to enrolling as a research scholar at Jawaharlal Nehru University, India, she received her graduate and post-graduate degrees in Biotechnology at Bangalore University, India. She has her expertise in the domain of molecular biology of the infectious disease mainly anthrax. She was a part of a team that worked towards developing therapeutic single chain variable fragment (Scfv) antibody against anthrax, the first of its kind. Her dedicated research on CodY, a pleiotropic transcriptional regulator, led to the revelation of the novel and unique aspects of this multifaceted protein. Further inquiry is being extended out from her to gain an insight into its detailed mechanism of interaction with GTP and further acquiring it as a drug target.