Role of W181 in modulating kinetic properties of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase

Sourav Roy, Tarak Karmakar, Lakshmeesha K. Nagappa, Vasudeva S. Prahlada Rao, Sundaram Balasubramanian, Hemalatha Balaram

Research output: Contribution to journalArticle

Abstract

Hypoxanthine-guanine-xanthine phosphoribosyltransference (HGXPRT), a key enzyme in the purine salvage pathway of the malarial parasite, Plasmodium falciparum (Pf), catalyses the conversion of hypoxanthine, guanine, and xanthine to their corresponding mononucleotides; IMP, GMP, and XMP, respectively. Out of the five active site loops (I, II, III, III', and IV) in PfHGXPRT, loop III' facilitates the closure of the hood over the core domain which is the penultimate step during enzymatic catalysis. PfHGXPRT mutants were constructed wherein Trp 181 in loop III' was substituted with Ser, Thr, Tyr, and Phe. The mutants (W181S, W181Y and W181F), when examined for xanthine phosphoribosylation activity, showed an increase in Km for PRPP by 2.1-3.4 fold under unactivated condition and a decrease in catalytic efficiency by more than 5-fold under activated condition as compared to that of the wild-type enzyme. The W181T mutant showed 10-fold reduced xanthine phosphoribosylation activity. Furthermore, molecular dynamics simulations of WT and in silico W181S/Y/F/T PfHGXPRT mutants bound to IMP.PPi.Mg2+ have been carried out to address the effect of the mutation of W181 on the overall dynamics of the systems and identify local changes in loop III'. Dynamic cross-correlation analyses show a communication between loop III' and the substrate binding site. Differential cross-correlation maps indicate altered communication among different regions in the mutants. Changes in the local contacts and hydrogen bonding between residue 181 with the nearby residues cause altered substrate affinity and catalytic efficiency of the mutant enzymes.

Original languageEnglish (US)
JournalProteins: Structure, Function and Bioinformatics
DOIs
StateAccepted/In press - 2016
Externally publishedYes

Keywords

  • Dynamic cross-correlations
  • Molecular dynamics simulations
  • PfHGXPRT
  • Phosphoribosyltransferase
  • Point mutation

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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