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

Important note: Information in this article was accurate in 1999. The state of the art may have changed since the publication date.

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

Nat Biotechnol. 1999 Mar;17(3):259-64. Unique Identifier : AIDSLINE MED/99193957
Christians FC; Scapozza L; Crameri A; Folkers G; Stemmer WP; Maxygen, Inc., Santa Clara, CA 95051, USA.

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 K(M) 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.

Keywords: JOURNAL ARTICLE Amino Acid Sequence Cell Division/DRUG EFFECTS Chimera Cloning, Molecular/METHODS Comparative Study Computer Simulation Dose-Response Relationship, Drug Escherichia coli/METABOLISM Herpesvirus 1, Human/*ENZYMOLOGY/GENETICS Herpesvirus 2, Human/*ENZYMOLOGY/GENETICS Human Hydrogen Bonding Kinetics Models, Molecular Molecular Sequence Data Mutagenesis Sequence Homology, Amino Acid Thymidine Kinase/GENETICS/METABOLISM/*THERAPEUTIC USE Zidovudine/METABOLISM/PHARMACOLOGY/*THERAPEUTIC USE
990930
A9990668

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