Designing a nucleotide-promiscuous FtsZ: An in vitro characterization

Abstract

For a cell to maintain its physiological optimum, biochemical reactions must occur with high efficiency and rapid kinetics. Enzymes are key in these and these enzymes often have an extreme specificity for a substrate. An example of such an enzyme is FtsZ, a key cytoskeletal protein essential for bacterial cell division, which exclusively utilizes GTP as its nucleotide substrate. However, the evolutionary reasons underlying the strict conservation of GTP as FtsZ’s native substrate remains unclear. To understand this, we aim to reengineer the nucleotide-binding pocket of FtsZ to accommodate ATP instead of GTP and investigate how this affects the kinetics and functioning of the enzyme and what physiological effects it imparts to the organism. In this study, we successfully engineered an ATPase-specific variant of FtsZ by combining a hypothetical GTP-binding defect mutation with a phosphate-binding motif mutation. Biochemical characterization of the mutant revealed significantly reduced GTPase activity, and diminished GTP-binding affinity, as shown by HPLC. Notably, the double mutant exhibited filament formation in the presence of ATP but failed to do so with GTP, strongly suggesting a change in its nucleotide specificity. In vivo studies are in progress to understand the impact of this ATPase mutant on cell division dynamics in Escherichia coli. These studies aim to elucidate the biological relevance of GTP specificity in the FtsZ mediated cell division process, providing fundamental insights into the evolutionary constraints for cell division to be regulated by GTP