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

Nature Biotechnology volume 17, pages 259–264 (1999)Cite this article

Abstract

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 ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, 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 acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced KM for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme–prodrug combinations.

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Figure 1: Titration of growth inhibition of TK-expressing KY895 cells by AZT.
Figure 2: (A) Shuffling of the two parental TK genes creates a library of chimeric genes.
Figure 3: (A) Minimized three-dimensional model of cycle 4 TK with bound ADP and dTMP showing the location of the five nonconserved amino acids (capped sticks).
Figure 4: Comparison of the hydrogen-bonding patterns of the binding sites of HSV-1 TK, HSV-2 TK, and cycle 4 TK with dTMP and AZT.

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Acknowledgements

Thanks to Sun-Ai Raillard, Glenn Dawes, and Steve delCardayre for technical assistance, to the Computational Center of the ETH Zürich for computer time, and to Claus Krebber, Larry Loeb, Frances Arnold, and Rolf Zinkernagel for comments on the manuscript.

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  1. Fred C. Christians

    Present address: Affymetrix, Inc., 3380 Central Expressway, Santa Clara, CA, 95051

Authors and Affiliations

  1. Maxygen, Inc., 3410 Central Expressway , Santa Clara, 95051, CA

    Fred C. Christians, Andreas Crameri & Willem P.C. Stemmer

  2. Department of Pharmacy, Swiss Federal Institute of Technology (ETH), Zürich, CH-8057, Switzerland

    Leonardo Scapozza & Gerd Folkers

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  1. Fred C. Christians
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Correspondence to Willem P.C. Stemmer.

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Christians, F., Scapozza, L., Crameri, A. et al. Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling. Nat Biotechnol 17, 259–264 (1999). https://doi.org/10.1038/7003

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