The International Journal of Biochemistry & Cell Biology 42 (2010) 253–262 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase Buddy Ullman a , Normand Cyr b , Kenneth Choi b , Armando Jardim b,∗ a Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97239, United States b Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, QC, Canada H9X 3V9 article info Article history: Received 10 June 2009 Received in revised form 8 October 2009 Accepted 16 October 2009 Available online 25 October 2009 Keywords: Phosphoribosyltransferase Purine metabolism HGPRT XPRT Leishmania abstract Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phospho- ribosyltransferase enzymes that mediate purine salvage, an obligatory nutritional function for these pathogenic parasites. The xanthine phosphoribosyltransferase preferentially uses xanthine as a substrate, while the hypoxanthine-guanine phosphoribosyltransferase phosphoribosylates only hypoxanthine and guanine. These related phosphoribosyltransferases were used as model system to investigate the molecular determinants regulating the 6-oxopurine specificity of these enzymes. Analysis of the purine binding domains showed two conserved acidic amino acids; glutamate residues in the xanthine phosphoribosyltransferase (E198 and E215) and aspartate residues in the hypoxanthine-guanine phos- phoribosyltransferase (D168 and D185). Genetic and biochemical analysis established that the single E198D and E215D mutations increased the turnover rates of the xanthine phosphoribosyltransferase without altering purine nucleobase specificity. However, the E215Q and E198,215D mutations converted the Leishmania xanthine phosphoribosyltransferase into a broad-specificity enzyme capable of utilizing guanine, hypoxanthine, and xanthine as substrates. Similarly, the D168,185E double mutation trans- formed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies estab- lished that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction Leishmania donovani, like all parasitic protozoa examined to date, lacks the capacity to synthesize purines de novo (Berman et al., 1991; Marr and Berens, 1983; Wang, 1984) and is dependent on metabolic pathways to scavenge nucleobases and nucleosides from the host. These pathways have attracted considerable attention as prospective chemotherapeutic targets (Wang, 1984; Berman et al., 1991; Marr and Berens, 1983; Martinez and Marr, 1992). Leishmania possess four purine salvage enzymes involved in converting preformed purine bases or nucleosides into nucleotides; xanthine phosphoribosyltransferase (LdXPRT), hypoxanthine- guanine phosphoribosyltransferase (LdHGPRT), adenine phospho- ribosyltransferase, and adenosine kinase. Genetic studies using conditional lethal ldhgprt/ldxprt double knockout cell lines revealed that a single copy either LdHGPRT or LdXPRT is necessary ∗ Corresponding author at: Institute of Parasitology, McGill University, 21, 111 Lakeshore Road, Ste-Anne-de-Bellevue, QC, Canada H9X 3V9. Tel.: +1 514 398 7727; fax: +1 514 398 7857. E-mail address: armando.jardim@mcgill.ca (A. Jardim). and sufficient to sustain parasite viability and infectivity (Boitz and Ullman, 2006). Purine phosphoribosyltransferases (PRTs) catalyze the Mg 2+ -dependent conversion of purine nucleobases and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nucleoside monophosphates and pyrophosphate (PP i ). There are distinct classes of 6-amino and 6-oxopurine PRTs. The 6-oxopurine PRTs in eukaryotes segregate into four groups based on nucleobase speci- ficity: (1) hypoxanthine-guanine-xanthine PRTs (HGXPRT) which are found in Toxoplasma gondii (Donald et al., 1996), Tritrichomonas foetus (Chin and Wang, 1994), and Plasmodium falciparum (Keough et al., 1999); (2) HGPRTs such as those found in mammals, Leish- mania, and other kinetoplastids (Allen et al., 1995; Allen et al., 1989; Giacomello and Salerno, 1978); (3) LdXPRT, a xanthine specific PRT unique to Leishmania (Jardim et al., 1999) and related trypanosomatids; and (4) a guanine phosphoribosyltransferase (GPRT) from Giardia lamblia (Page et al., 1999). Structures of HGPRTs and HGXPRTs in the apoenzyme form, as well as ternary transition-state and nucleotide product-bound complexes have been reported (Heroux et al., 1999a,b, 2000; Focia et al., 1998a; Eads et al., 1994; Balendiran et al., 1999; Keough et al., 2005; Monzani et al., 2007; Shi et al., 1999; Schumacher et al., 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.10.020