Unusual substrate specificity of a chimeric hypoxanthine-guanine phosphoribosyltransferase containing segments from the Plasmodium falciparum and human enzymes.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the phosphoribosylation of hypoxanthine and guanine by transferring the phosphoribosyl moiety from phosphoribosylpyrophosphate (PRPP) on to N9 in the purine base, resulting in the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP). Xanthine is an additional substrate for the Plasmodium falciparum HGXPRT. Our aim has been to elucidate structural features in HGPRT that govern substrate specificity. We have addressed this problem by engineering chimeric HGPRTs, which contain segments from both the parasite and human enzymes. Four chimeric enzymes were engineered (DS1-DS4), of which the chimeric enzyme, DS1, in which the first 49 residues of human HGPRT were replaced with the corresponding residues from the P. falciparum enzyme, exhibited additional specificity for xanthine. None of the switched residues forms a part of the purine or PRPP binding region in the available crystal structures of HG(X)PRTs. Our data on the chimeric enzyme DS1 provide the first evidence that the N-terminal approximately 50 amino acids, although not proximal to the active site in the crystal structure, can in fact modulate substrate specificity. DS1 exhibits a reduced k(cat) for hypoxanthine and guanine, while its K(m) for these oxopurine bases remains largely unchanged. Its specific activity for xanthine is comparable with hypoxanthine but five times more than that for guanine.
Sujay Subbayya
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Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.
Sukumaran
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Shivashankar
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Balaram
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000254:growth & development; D006147:Guanine / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D019820:Xanthine / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D019820:Xanthine; D019271:Hypoxanthine; D006147:Guanine; D010754:Phosphoribosyl Pyrophosphate; D006157:Guanosine Monophosphate; D007041:Hypoxanthine Phosphoribosyltransferase