Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation.

Lionel Ballut,Raphaël Terreux,Nushin Aghajari,Lakshmi Prasoona Thota,Manu Sathya,Hemalatha Balaram,Bauke W Dijkstra,Jyothirmai Kunala,Sébastien Violot,Santosh Shivakumaraswamy,Richard Haser

doi:10.1038/ncomms9930

Abstract

GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371–375 holding catalytic residues and in loop 376–401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk. These studies reveal the mechanism of domain rotation and inter-domain communication, providing a molecular framework for the function of all single polypeptide GMPSs and form a solid basis for rational drug design targeting this therapeutically important enzyme.

Highlights

guanosine monophosphate (GMP) synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear
During the parasite’s intra-erythrocytic stages, adenosine and hypoxanthine salvaged from the human host serve as precursors for synthesizing inosine monophosphate (IMP), which serves as precursor for the synthesis of both adenosine monophosphate (AMP) and guanosine monophosphate (GMP)
GMP synthesis proceeds through two steps, the first being the conversion of IMP to xanthine monophosphate (XMP) catalysed by IMP dehydrogenase followed by GMP synthetase (GMPS) converting XMP to GMP4

Summary

Results

All three mutants exhibited activation of GATase activity upon ligandbinding to the ATPPase domain, PfGMPS_W167F compared with the wild-type enzyme showed 60% reduction in leaky GATase activity (Fig. 4c) These differences suggest that interactions seen in the crystal structure, involving these three residues, are important for at least the structural integrity of the GATase active site and thereby do have functional implications. The domain rotation reorganizes the aromatic cluster at the interface of the two domains resulting in a new interaction between Tyr[212] (GATase domain) and Tyr[369] (ATPPase domain) (Fig. 3d) While both kcat and Km values for NH4Cl for PfGMPS_Y212W did not show any significant change, the Km value for Gln showed a 2.8-fold increase with a 2-fold drop in kcat/Km when compared with PfGMPS (Table 2), again corroborating the structural observations.

F32 F235 Y240

Discussion

H20 E374– D371 b

Methods