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- Slot Christiansen, Louise, et al.
(författare)
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New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine.
- 2015
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 7:2, s. 966-980
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Tidskriftsartikel (refereegranskat)abstract
- Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT).We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.
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| 3. |
- Egeblad, Louise
(författare)
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Structural and functional studies of enzymes in nucleotide metabolism : a detailed investigation of two enzymes and interaction profiling of FDA-approved nucleoside analog drugs with 23 enzymes
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Enzymes in nucleotide metabolism serve as the producers of the building blocks for DNA and RNA. From a medical perspective, nucleotide metabolism, and in particular salvage pathway enzymes, have attracted special interest, as nucleoside prodrugs given in the treatment of cancer and HIV are converted into their active metabolite forms by these enzymes. In this thesis, two enzymes; uridine monophosphate kinase (UMPK) from Ureaplasma parvum (Up) and human phosphoribosyltransferase domain containing protein 1 (PRTFDC1), have been investigated. Furthermore, a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs has been developed. The structure of Up-UMPK revealed that it was a hexamer. Kinetic constants were determined for UMP and ATP. UTP was a competitive inhibitor of UMP, and a non-competitive inhibitor of ATP. In contrast to other bacterial UMPKs, Up-UMPK was not activated by GTP. PRTFDC1 is a homolog of hypoxanthine-guanine phosphoribosyltransferase (HPRT). Mutations in HPRT are associated with Lesch-Nyhan syndrome. The three-dimensional structures of PRTFDC1 and HRPT are very similar. Even though PRTFDC1 recognizes guanine and hypoxanthine as substrates, the functional turnover rates are less than 1% of the activity of HPRT. NAL was screened using the high-throughput method, differential static light scattering (DSLS). An interaction profile of 23 enzymes involved in nucleotide metabolism and NAL was revealed. Interactions were detected for uridine phosphorylase 1 (UPP1) and guanine deaminase (GDA) with eight and six nucleoside prodrugs, respectively. The knowledge gained from this study can be important in the future search for drug lead candidates for UPP1 and GDA.
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| 4. |
- Egeblad, Louise, et al.
(författare)
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Structural and functional studies of the human phosphoribosyltransferase domain containing protein 1
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:23, s. 4920-30
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Tidskriftsartikel (refereegranskat)abstract
- Human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (EC 2.4.2.8) 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 (k(cat)/K(m)) 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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