Ammonia Channeling in <i>Plasmodium falciparum</i> GMP Synthetase: Investigation by NMR Spectroscopy and Biochemical Assays

Bhat, J.Y.; Venkatachala, Roopa; Singh, Kavita; Gupta, K. P.; Sarma, Siddhartha P.; Balaram, Hemalatha

doi:10.1021/bi1017057

Cited by 16 publications

(49 citation statements)

References 46 publications

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“…Also, the crystal structures do not reveal an ammonia tunnel connecting the two active sites [22,27]. Regardless of the distal arrangement of active sites and the absence of a tunnel, biochemical studies provide evidence for regulation of GATase activity by the binding of substrates to the acceptor domain and ammonia tunnelling [28,29]. Our recent crystal structure of P. falciparum GMPS (PfGMPS) has revealed that an 85° rotation of the GATase domain ( Fig.…”

Section: Introductionmentioning

confidence: 98%

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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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Section: Introductionmentioning

confidence: 98%

“…The functioning of PfGMPS has been addressed by biochemical experiments and crystal structures [28][29][30]. The enzyme catalyses the conversion of XMP to GMP with the catalytic event in the acceptor domain (ATP pyrophosphatase domain (ATPPase)) being the synthesis of adenyl-XMP intermediate from ATP.Mg 2+ and XMP (Fig.…”

Section: Introductionmentioning

confidence: 99%

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

Shivakumaraswamy

Pandey

Ballut

et al. 2019

Preprint

Self Cite

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“…Using distinct approaches, similar observations were recently made for the P. falciparum form of the enzyme (33); additional insights regarding glutaminase activation and ammonia utilization have also been recently gained for this enzyme (34). These studies provided evidence that GMPS can utilize exogenous ammonia in the presence of glutamine, while the exchange of glutamine-derived ammonia with bulk solvent was not observed, indicating the fidelity of the coupled catalytic processes.…”

Section: Introductionmentioning

confidence: 69%

Substrate Activation and Conformational Dynamics of Guanosine 5′-Monophosphate Synthetase

et al. 2013

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Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. GMP synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of XMP. In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid ammonia transfer to this intermediate. Isolation and use of the adenylylated nucleotide intermediate enabled study of the amidotransfer reaction distinct from the ATP dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear faster than steady-state turnover, implying that release is likely rate limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.

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“…The C-terminal domain is an ATP pyrophosphatase domain, where the adenyl–XMP intermediate is formed [112]. The substrate-protective environment created for the channeling of ammonia between the two separate although connected GMPS domains ensures an efficient catalysis and classifies this enzyme as a Class I amidotransferase [113–115]. The strict requirement for selective toxicity for inhibitors directed towards enzymes in the purine salvage pathway evident in cases like HGXPRT, where co-inhibition of the human homolog has pathologic consequences [83–85], may not apply to GMPS.…”

Section: Purine Salvage Enzymes and Transition-state Analogsmentioning

confidence: 99%

Transition-State Inhibitors of Purine Salvage and Other Prospective Enzyme Targets in Malaria

2013

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Malaria is a leading cause of human death within the tropics. The gradual generation of drug resistance imposes an urgent need for the development of new and selective antimalarial agents. Kinetic isotope effects coupled to computational chemistry have provided the relevant details on geometry and charge of enzymatic transition states to facilitate the design of transition-state analogs. These features have been reproduced into chemically stable mimics through synthetic chemistry, generating inhibitors with dissociation constants in the pico- to femto-molar range. Transition-state analogs are expected to contribute to the control of malaria.

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Ammonia Channeling in Plasmodium falciparum GMP Synthetase: Investigation by NMR Spectroscopy and Biochemical Assays

Cited by 16 publications

References 46 publications

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

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

Substrate Activation and Conformational Dynamics of Guanosine 5′-Monophosphate Synthetase

Transition-State Inhibitors of Purine Salvage and Other Prospective Enzyme Targets in Malaria

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