| 1. |
- Christoffersen, Stig, et al.
(författare)
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Nucleoside phosphorylases from clostridium perfringens in the synthesis of 2',3'-dideoxyinosine.
- 2010
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Ingår i: Nucleosides, Nucleotides & Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 29:4-6, s. 445-448
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Tidskriftsartikel (refereegranskat)abstract
- Four Clostridium perfringens phosphorylases were subcloned, overexpressed and analyzed for their substrate specificity. DeoD(1) and PunA could use a variety of purine substrates, including an antiviral drug 2',3'-dideoxyinosine (ddI). In one-pot synthesis using Clostridium phosphorylases, 2',3'-dideoxyuridine and hypoxanthine were converted to ddI at yield of about 30%.
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| 2. |
- Serra, Immacolata, et al.
(författare)
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Developing a Collection of Immobilized Nucleoside Phosphorylases for the Preparation of Nucleoside Analogues: Enzymatic Synthesis of Arabinosyladenine and 2,3-Dideoxyinosine
- 2013
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Ingår i: Collection of Czechoslovak Chemical Communications. - : Wiley. - 2192-6506. ; 78:2, s. 157-165
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Tidskriftsartikel (refereegranskat)abstract
- The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly expressed nucleoside phosphorylases from the bacterial pathogens Citrobacter koseri, Clostridium perfringens, and Streptococcus pyogenes (CkPNPI, CkPNPII, CpUP, SpUP) and their substrate specificity was investigated towards either natural pyrimidine or purine nucleosides and some analogues, namely, arabinosyladenine (araA) and 2,3-dideoxyinosine (ddI). A 23% activity towards these latter compounds (compared to the natural substrates) was observed. Enzyme activities were compared to the specificities obtained for the enzymes pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP) and purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNPII) previously reported by some of the authors. The enzymes displaying the suitable specificity for the synthesis of araA and ddI were immobilized on aldehydeagarose. The immobilized preparations were highly stable at alkaline pH and in the presence of methanol or acetonitrile as cosolvent. They were used in the synthesis of araA and ddI by a one-pot, bienzymatic transglycosylation achieving 74 and 44% conversion, respectively.
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| 3. |
- Tran, Timothy H., et al.
(författare)
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The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases
- 2011
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 50:30, s. 6549-6558
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Tidskriftsartikel (refereegranskat)abstract
- Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose I-phosphate and uracil, at 1.8 angstrom resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an alpha/beta monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUTP showed that its substrate specificity is similar to that of EcUP, while EcUP is similar to 7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His 169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.
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