The Conserved Serine-Tyrosine Dipeptide in Leishmania donovani Hypoxanthine-guanine Phosphoribosyltransferase Is Essential for Catalytic Activity

Abstract

Protozoan parasites constitute a devastating public health and socioeconomic burden in the tropical and subtropical regions of the world. In the absence of effective vaccines, empirically derived drugs are the only means available to treat parasitic infections. However, the current arsenal of antiparasitic drugs is far from ideal, as these agents are moderately to highly toxic, possibly mutagenic and/or carcinogenic, and require protracted treatment regimens. The control of parasitic diseases has also been complicated by the emergence of drugresistant strains (1), further underscoring the need for novel antiparasitic agents. Rational development of new drugs that selectively target protozoan pathogens requires the exploitation of biochemical or metabolic differences between parasite and host. As protozoan parasites, unlike mammalian cells, are auxotrophic for purines and consequently rely on purine appropriation from the host for survival and proliferation (2), the purine salvage pathway has stimulated considerable interest as a paradigm for antiparasitic drug development. One enzyme that is central to purine acquisition in many parasites (2) is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) 1 which catalyzes the phosphoribosylpyrophosphate (PRPP)-dependent phosphoribosylation of hypoxanthine and guanine to the nucleotide level. In some parasites, HGPRT also recognizes xanthine as a substrate (3) and is, therefore, termed a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGX-PRT). Genes and cDNAs encoding HG(X)PRT (HG(X)PRT) from a number of protozoan parasites have now been cloned, sequenced, and overexpressed in Escherichia coli, and the recombinant HG(X)PRT enzymes have been purified and characterized kinetically (3). Multisequence alignments demonstrate several short regions of amino acid homology among HG(X)-PRT family members that are flanked by much longer regions without significant sequence similarity. The crystal structures for the product-bound form of the human HGPRT (4) and the Tritrichomonas foetus HGXPRT (5) have revealed, however, a common core framework within the tertiary structure that is conserved among other members of the phosphoribosyltransferase (PRT) family (6 -8). These structures have established functional roles for all of the conserved regions in HG(X)PRT proteins, except one motif that is located on a flexible loop distal to the active site. This flexible loop encompasses a SerTyr dipeptide that is found in all HG(X)PRTs for which sequence is available. The recently determined crystal structures of the HGXPRT from Toxoplasma gondii in the absence of ligand and in the presence of product have demonstrated that this flexible loop in the apoenzyme is shifted toward the active

Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis

“…3). This mechanism of catalysis is identical to all the other purine PRTase‐catalyzed reactions reported thus far [18, 23, 24]. The K ii and K is values from the product inhibition studies are summarized in Table 2.…”

“…The ratio of forward/reverse V max is calculated to be 13.4, which demonstrates a clear bias towards GMP synthesis. Furthermore, despite a relatively high K m for guanine at 16.4 µ m , its catalytic efficiency for guanine ( k cat / K m = 4.7 µ m −1 s −1 ) was higher than the catalytic efficiencies for guanine in other HG(X)PRTase‐catalyzed reactions (1.9 µ m −1 s −1 for S. mansoni HGPRTase [22]; 3.7 µ m −1 s −1 for human HGPRTase [23]; 1.0 µ m −1 s −1 for T. foetus HGXPRTase [18] but lower than that of 8.1 µ m −1 s −1 for Leishmania donovani HGPRTase [24]. A previous report on a lower catalytic rate for the recombinant Giardia GPRTase [4] was attributed to an inadvertent mutation of D129G in the encoding gene while it was synthesized by PCR and ligated into the expression vector.…”

Xanthine Phosphoribosyltransferase from Leishmania donovani

“…The substrate specificities (and kinetic parameters) ascribed to both the purified recombinant L. donovani XPRT (Fig. 8) and HGPRT (15,21) are supported by the results of the complementation analyses in which only efficient substrates could support the growth of S⌽609 transformants in minimal medium.…”

Purine Phosphoribosyltransferases

“…Analysis of the HPRT from L. donovani revealed that mutations of the residue homologous with the tyrosine at position 104 of the human HPRT severely reduce the turnover number (k cat ) for the forward reaction catalyzed by the enzyme (33). The main chain nitrogen of Tyr-104 forms a hydrogen bond with a pyrophosphate oxygen (Fig.…”