Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase

Abstract

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate 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. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

Mechanistic insights into the functioning of GMP synthetase: a two-subunit, allosterically regulated, ammonia tunnelling enzyme

“…The dimerization domain is also important for ligand binding as residues on a loop at the C-terminal extreme and inter-dimer interactions of a conserved Arg residue are crucial for binding XMP. 28 In MjATPPase/XMP structure, the interactions of the residues on the C-terminal loop (residues 298-310) with XMP as well as the inter-dimer interactions of Arg294 are conserved (Figure 4B).…”

“…The corresponding residues in PfGMPS play roles in catalysing the synthesis of AMP-XMP intermediate and GMP, respectively. 28 Considering that His145 and Asn146 are positioned > 13 Å away from the bound XMP, the conformation of the lid-loop in MjATPPase must correspond to an inactive or resting state of the enzyme. Thus, subsequent to the binding of ATP.Mg 2+ +XMP, conformational changes in the lid-loop must reposition His145 and Asn146 in proximity to the substrates.…”

“…The amino acid sequences of the interface helices are highly conserved, and the conserved sequence motifs are enriched with charged residues (Figure S9A). 28 Further, residues of the conserved motif of GATase helix α1 form inter-domain salt-bridge interactions with residues on the ATPPase helices α11 and α12 motifs (Figure S9B). Perturbing these inter-domain interactions in PfGMPS by mutagenesis revealed that the α1-α12 inter-domain salt-bridge interactions are conduits for information exchange pertaining to allostery through which the catalytic events in the ATPPase domain are communicated to the GATase domain.…”

“…Perturbing these inter-domain interactions in PfGMPS by mutagenesis revealed that the α1-α12 inter-domain salt-bridge interactions are conduits for information exchange pertaining to allostery through which the catalytic events in the ATPPase domain are communicated to the GATase domain. 28…”

The Adaptive Evolution and Gigantism Mechanisms of the Hadal “Supergiant” Amphipod Alicella gigantea

“…Moreover, IMPDH1 (PSG as shown in Figure 5A and Table 2) is involved in nucleotide metabolism by converting IMP to XMP (Slee and Bownes, 1995). XMP can be further converted to GMP (Shivakumaraswamy et al, 2020), which can promote the growth performance of organisms (Hossain et al, 2016). Therefore, we can conclude that the two PSGs (ITPK and IMPA2) are involved in the inositol phosphate metabolism pathway, and together with IMPDH1 would ultimately be related to growth and proliferations in A. gigantea.…”