Structural and function studies of the human phosphoribosyltransferase domain containing protein 1

Structural Genomics Consortium, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
FEBS Journal (Impact Factor: 4). 10/2010; 277(23):4920-30. DOI: 10.1111/j.1742-4658.2010.07897.x
Source: PubMed


Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC catalyzes the conversion of hypoxanthine and guanine to their respective nucleoside monophosphates. Human HPRT deficiency as a result of genetic mutations is linked to both Lesch–Nyhan disease and gout. In the present study, we have characterized phosphoribosyltransferase domain containing protein 1 (PRTFDC1), a human HPRT homolog of unknown function. The PRTFDC1 structure has been determined at 1.7 Å resolution with bound GMP. The overall structure and GMP binding mode are very similar to that observed for HPRT. Using a thermal-melt assay, a nucleotide metabolome library was screened against PRTFDC1 and revealed that hypoxanthine and guanine specifically interacted with the enzyme. It was subsequently confirmed that PRTFDC1 could convert these two bases into their corresponding nucleoside monophosphate. However, the catalytic efficiency (kcat/Km) of PRTFDC1 towards hypoxanthine and guanine was only 0.26% and 0.09%, respectively, of that of HPRT. This low activity could be explained by the fact that PRTFDC1 has a Gly in the position of the proposed catalytic Asp of HPRT. In PRTFDC1, a water molecule at the position of the aspartic acid side chain position in HPRT might be responsible for the low activity observed by acting as a weak base. The data obtained in the present study indicate that PRTFDC1 does not have a direct catalytic role in the nucleotide salvage pathway.
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Available from: Pål Stenmark, Sep 28, 2014
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    • "To further investigate the mechanism by which 6-TG inhibited Mpn growth, Mpn HPRT was expressed, purified, and characterized. Both Hx and Gua are good substrates for the enzyme and the Vmax values for these substrates are in the same order of magnitude as the human enzyme [44]. In humans, the plasma concentrations of Hx and Gua are approximately 172 μM and 97 μM [45], which is close to the Km and S0.5 values of Mpn HPRT with Hx and Gua. "
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    ABSTRACT: Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates. Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 mug ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 mug ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources. We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.
    BMC Microbiology 08/2013; 13(1):184. DOI:10.1186/1471-2180-13-184 · 2.73 Impact Factor
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    • "First, since there is unvalidated evidence that the HPRT and PRTFDC1 proteins may physically interact with one another [15], the regulation, or activity of PRTFDC1 might be altered when HPRT is absent or mutated. Second, though it is not known if PRTFDC1 has biologically relevant phosphoribosyltransferase activity [16], because the activity of other phosphoribosyltransferases with known enzymatic activity has been reported to be increase in the absence of HPRT due to elevated levels of a common substrate, 5-phosphoribosyl-1-pyrophosphate (PRPP) , reviewed in [4], the activity of PRTFDC1 has the potential to be similarly affected. We therefore proposed that when HPRT is absent or mutated, the altered activity and/or protein-protein interactions of PRTFDC1 enhance the phenotypic severity of HPRT-deficiency. "
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    ABSTRACT: Lesch-Nyhan disease (LND) is a severe X-linked neurological disorder caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT). In contrast, HPRT-deficiency in the mouse does not result in the profound phenotypes such as self-injurious behavior observed in humans, and the genetic basis for this phenotypic disparity between HPRT-deficient humans and mice is unknown. To test the hypothesis that HPRT deficiency is modified by the presence/absence of phosphoribosyltransferase domain containing 1 (PRTFDC1), a paralog of HPRT that is a functional gene in humans but an inactivated pseudogene in mice, we created transgenic mice that express human PRTFDC1 in wild-type and HPRT-deficient backgrounds. Male mice expressing PRTFDC1 on either genetic background were viable and fertile. However, the presence of PRTFDC1 in the HPRT-deficient, but not wild-type mice, increased aggression as well as sensitivity to a specific amphetamine-induced stereotypy, both of which are reminiscent of the increased aggressive and self-injurious behavior exhibited by patients with LND. These results demonstrate that PRTFDC1 is a genetic modifier of HPRT-deficiency in the mouse and could therefore have important implications for unraveling the molecular etiology of LND.
    PLoS ONE 07/2011; 6(7):e22381. DOI:10.1371/journal.pone.0022381 · 3.23 Impact Factor
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