Design and directed evolution of a dideoxy purine nucleoside phosphorylase.
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.
Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
David P
DP
Nannemann
Kristian W
KW
Kaufmann
Jens
J
Meiler
Brian O
BO
Bachmann
D019943:Amino Acid Substitution; D016049:Didanosine / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012984:Software; D013816:Thermodynamics
D011683:Purine-Nucleoside Phosphorylase; D016049:Didanosine