Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high throughput robotic screen identified chimeras with an enhanced abilit y to phosphoryl;ate zidovudine (AZT). Improved clones were combined, reshuf fled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino a cid substitutions not found in either parent, including active site mutatio ns. Kinetic measurements show that the chimeric enzymes had acquired reduce d Kw for AZT as well as decreased specificity for thymidine. In agreement w ith the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expen se of thymidine. Despite the overall similarity of the two chimeric enzymes , each contains key contributions from different parents in positions influ encing substrate affinity. Such mutants could be useful for anti-HIV gene t herapy, and similar directed-evolution approaches could improve other enzym e-prodrug combinations.