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

• Published in: Proteins, structure, function, and bioinformatics Vol. 84; no. 11; pp. 1658 - 1669
• Main Authors: Roy, Sourav, Karmakar, Tarak, Nagappa, Lakshmeesha K., Prahlada Rao, Vasudeva S., Balasubramanian, Sundaram, Balaram, Hemalatha
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.11.2016; Wiley Subscription Services, Inc
• Subjects: Amino Acid Substitution; Catalytic Domain; dynamic cross-correlations; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Hydrogen Bonding; Inosine Monophosphate - chemistry; Inosine Monophosphate - metabolism; Kinetics; Molecular Dynamics Simulation; molecular dynamics simulations; Mutation; Pentosyltransferases - chemistry; Pentosyltransferases - genetics; Pentosyltransferases - metabolism; PfHGXPRT; Phenylalanine - chemistry; Phenylalanine - metabolism; phosphoribosyltransferase; Plasmodium falciparum; Plasmodium falciparum - chemistry; Plasmodium falciparum - enzymology; point mutation; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Serine - chemistry; Serine - metabolism; Structure-Activity Relationship; Substrate Specificity; Thermodynamics; Threonine - chemistry; Threonine - metabolism; Tryptophan - chemistry; Tryptophan - metabolism; Tyrosine - chemistry; Tyrosine - metabolism; molecular dynamics simulations; phosphoribosyltransferase; dynamic cross-correlations; point mutation; PfHGXPRT
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.25107

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. Proteins 2016; 84:1658–1669. © 2016 Wiley Periodicals, Inc.