Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase

Oliver, Justin C.; Gudihal, Ravidra; Burgner, John W.; Pedley, Anthony M.; Zwierko, Alexander T.; Davisson, V. Jo; Linger, Rebecca S.

doi:10.1016/j.abb.2014.01.004

Cited by 11 publications

(11 citation statements)

References 67 publications

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“…As no clear channel for ammonia transfer emerges from these structures, a drastic structural reorientation could be expected in a fully liganded enzyme. This view was recently suggested by biochemical studies of Ec GMPS 13 .…”

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confidence: 73%

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

et al. 2015

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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.

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confidence: 73%

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

et al. 2015

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“…Mutation of His186 to Ala resulted in an increase in the Km value for Gln by 50-fold and uncoupling of the two reactions [32] Interface helix residues of the ATPPase domain are also involved in catalysis Residues Arg25 on α1, Asp371 on α11, and Lys411 and Lys415 on α12 are either invariant or conservatively replaced but have side chains directed away from the interdomain interface ( Fig. 3a and 5).…”

Section: Interdomain Interactions In the 85° Interface Also Modulate mentioning

confidence: 99%

Helices on interdomain interface couple catalysis in the ATPPase domain with allostery inPlasmodium falciparumGMP synthetase

Shivakumaraswamy

Pandey

Ballut

et al. 2019

Preprint

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AbstractGMP 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 α11 and α12 that are located at the interdomain interface, and the lid-loop that follows α11. This apart, perturbing interdomain interactions involving residues on α11 and α12 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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“…S1). Recently, the mechanism of activation of GATs in GMPS enzyme has been investigated by different groups and they suggest that subunit interfaces may play an important role in GATase activation . However, site directed mutagenesis study of carbamoyl phosphate synthetase in Escherichia coli indicates some catalytic role of Asn311 and Ser47 in GAT domain .…”

Section: Introductionmentioning

confidence: 99%

New insight into the architecture of oxy‐anion pocket in unliganded conformation of GAT domains: A MD‐simulation study

Bairagya

Bansal

2016

Proteins

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Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N-terminal "glutaminase" (GAT) and C-terminal "synthetase" domain. The enzyme is identified as a potential target for anti-cancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H-bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)-substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non-catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site.

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Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase

Cited by 11 publications

References 67 publications

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

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

Helices on interdomain interface couple catalysis in the ATPPase domain with allostery inPlasmodium falciparumGMP synthetase

New insight into the architecture of oxy‐anion pocket in unliganded conformation of GAT domains: A MD‐simulation study

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