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bioRxiv · 10.1101/808618

Interactions of helices on interdomain interface couple the chemistry of intermediate formation with allostery in Plasmodium falciparum GMP synthetase

Shivakumaraswamy, S.·Pandey, N.·Ballut, L.·Violot, S.·Aghajari, N.·Balaram, H.
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Abstract

GMP synthetase catalyzes the substitution of the C2 oxo-group of the purine base in XMP with an amino-group generating GMP, the last step in the biosynthesis of GMP. This reaction involves a series of catalytic events that include hydrolysis of Gln generating ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. Inherent to the functioning of GMP synthetases is bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain by substrates binding to the ATPPase domain, synchronization of the two catalytic events and tunnelling of ammonia from the GATase to the ATPPase domain. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and, steady-state and transient kinetic assays on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. The results map the residues critical for catalysis in the ATPPase domain to the helices 11 and 12 that are located at the interdomain interface, and the lid-loop that follows 11. This apart, perturbing interdomain interactions involving residues on 11 and 12 impairs GATase activation. These results imply that this arrangement of helices at the domain interface with residues that play roles in ATPPase catalysis as well as domain crosstalk enables coupling ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

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Shivakumaraswamy, S., Pandey, N., Ballut, L., Violot, S., Aghajari, N., Balaram, H.. 2019-10-23. Interactions of helices on interdomain interface couple the chemistry of intermediate formation with allostery in Plasmodium falciparum GMP synthetase. https://doi.org/10.1101/808618

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