ArticleProtein engineering, design & selection : PEDS2010
Design and directed evolution of a dideoxy purine nucleoside phosphorylase.
Article in Protein engineering, design & selection : PEDS, 2010. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 33 citations in OpenAlex.
- Biocatalytic Synthesis of Antiviral Nucleosides, Cyclic Dinucleotides, and Oligonucleotide Therapies.JACS Au · 2023Review
- The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a `spanner in the works'.Acta crystallographica. Section F, Structural biology communications · 2022Article
- Bioretrosynthetic construction of a didanosine biosynthetic pathway.Nature chemical biology · 2014Article
- Small-molecule ligand docking into comparative models with Rosetta.Nature protocols · 2013Article
- Molecular differences between a mutase and a phosphatase: investigations of the activation step in Bacillus cereus phosphopentomutase.Biochemistry · 2012Article
- Analysis ofJournal of pharmaceutical analysis · 2012Article
- Computational approaches for rational design of proteins with novel functionalities.Computational and structural biotechnology journal · 2012Review
- Article
- Assessing directed evolution methods for the generation of biosynthetic enzymes with potential in drug biosynthesis.Future medicinal chemistry · 2011Article
- Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a purine nucleoside phosphorylase from Bacillus subtilis strain 168.Acta crystallographica. Section F, Structural biology and crystallization communications · 2011Article
- Computational design of protein-ligand interfaces: potential in therapeutic development.Trends in biotechnology · 2011Review
- Bacillus cereus phosphopentomutase is an alkaline phosphatase family member that exhibits an altered entry point into the catalytic cycle.The Journal of biological chemistry · 2011Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
Purine nucleoside phosphorylase (PNP) catalyzes the synthesis and phosphorolysis of purine nucleosides, interconverting nucleosides with their corresponding purine base and ribose-1-phosphate. While PNP plays significant roles in human and pathogen physiology, we are interested in developing PNP as a catalyst for the formation of nucleoside analog drugs of clinical relevance. Towards this aim, we describe the engineering of human PNP to accept 2',3'-dideoxyinosine (ddI, Videx((R))) as a substrate for phosphorolysis using a combination of site-directed mutagenesis and directed evolution. In human PNP, we identified a single amino acid, Tyr-88, as a likely modulator of ribose selectivity. RosettaLigand was employed to calculate binding energies for substrate and substrate analog transition state complexes for single mutants of PNP where Tyr-88 was replaced with another amino acid. In parallel, these mutants were generated by site-directed mutagenesis, expressed and purified. A tyrosine to phenylalanine mutant (Y88F) was predicted by Rosetta to improve PNP catalytic activity with respect to ddI. Kinetic characterization of this mutant determined a 9-fold improvement in k(cat) and greater than 2-fold reduction in K(M). Subsequently, we used directed evolution to select for improved variants of PNP-Y88F in Escherichia coli cell extracts resulting in an additional 3-fold improvement over the progenitor strain. The engineered PNP may form the basis for catalysts and pathways for the biosynthesis of ddI.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.