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.
Nannemann
David P
DP
Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
Kaufmann
Kristian W
KW
Meiler
Jens
J
Bachmann
Brian O
BO
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