Acidic residues in the purine binding site govern the 6-oxopurine specificity of the Leishmania donovani xanthine phosphoribosyltransferase.
Leishmania possess distinct xanthine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase 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 phosphoribosyltransferase (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 transformed the Leishmania hypoxanthine-guanine phosphoribosyltransferase into a mutant enzyme capable phosphoribosylating only xanthine, albeit with a much lower catalytic efficiency. These studies established that these conserved acidic residues play an important role in governing the nucleobase selectivity of the Leishmania 6-oxopurine phosphoribosyltransferases.
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97239, USA.
Buddy
B
Ullman
Normand
N
Cyr
Kenneth
K
Choi
Armando
A
Jardim
D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D006157:Guanosine Monophosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011688:Purinones / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D011688:Purinones; D008345:Manganese; D006157:Guanosine Monophosphate; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D008274:Magnesium