Directed evolution of an orthogonal nucleoside analog kinase via fluorescence-activated cell sorting

Nucleoside analogs (NAs) represent an important category of prodrugs for the treatment of viral infections and cancer, yet the biological potency of many analogs is compromised by their inefficient activation through cellular 2′-deoxyribonucleoside kinases (dNKs). We herein report the directed evolution and characterization of an orthogonal NA kinase for 3′-deoxythymidine (ddT), using a new FACS-based screening protocol in combination with a fluorescent analog of ddT. Four rounds of random mutagenesis and DNA shuffling of Drosophila melanogaster 2′-deoxynucleoside kinase, followed by FACS analysis, yielded an orthogonal ddT kinase with a 6-fold higher activity for the NA and a 20-fold kcat/KM preference for ddT over thymidine, an overall 10 000-fold change in substrate specificity. The contributions of individual amino acid substitutions in the ddT kinase were evaluated by reverse engineering, enabling a detailed structure–function analysis to rationalize the observed changes in performance. Based on our results, kinase engineering with fluorescent NAs and FACS should prove a highly versatile method for evolving selective kinase:NA pairs and for studying fundamental aspects of the structure–function relationship in dNKs.

[1]  Andrew G. Watts,et al.  Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis , 2006, Nature chemical biology.

[2]  G. Georgiou.  Analysis of large libraries of protein mutants using flow cytometry. , 2000, Advances in protein chemistry.

[3]  S. Lutz,et al.  Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2. , 2007, Biochemical and biophysical research communications.

[4]  G. Gentry.  Viral thymidine kinases and their relatives. , 1992, Pharmacology & therapeutics.

[5]  E. De Clercq,et al.  The anti-HTLV-III (anti-HIV) and cytotoxic activity of 2',3'-didehydro-2',3'-dideoxyribonucleosides: a comparison with their parental 2',3'-dideoxyribonucleosides. , 1987, Molecular pharmacology.

[6]  T. Kunkel,et al.  DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[7]  K. Müller,et al.  Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution , 2005, Nucleic acids research.

[8]  D. Bergstrom,et al.  Pyrido(2,3-d)pyrimidine Nucleosides. Synthesis via Cyclization of C-5-Substituted Cytidines , 1982 .

[9]  L. Loeb,et al.  Thymidine kinase mutants obtained by random sequence selection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Z. Ram,et al.  In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. , 1992, Science.

[11]  J. Barrio,et al.  Fluorescent adenosine and cytidine derivatives. , 1972, Biochemical and biophysical research communications.

[12]  C. Monnerjahn,et al.  Binding of ATP to TK1-like enzymes is associated with a conformational change in the quaternary structure. , 2007, Journal of molecular biology.

[13]  C. Cass,et al.  Studies of nucleoside transporters using novel autofluorescent nucleoside probes. , 2006, Biochemistry.

[14]  K. Igarashi,et al.  A deoxythymidine kinase deficient mutant of Escherichia coli. II. Mapping and transduction studies with phage phi 80. , 1967, Genetics.

[15]  H. Eklund,et al.  Structures of thymidine kinase 1 of human and mycoplasmic origin , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  R. G. Hughes,et al.  Effects of 2′,3′‐dideoxynucleosides on mammalian cells and viruses , 1984, Journal of cellular physiology.

[17]  S. Lutz,et al.  Systematic exploration of active site mutations on human deoxycytidine kinase substrate specificity. , 2008, Biochemistry.

[18]  J. Piškur,et al.  Functional Expression of a Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster and Its C-terminal Deletion Mutants* , 2000, The Journal of Biological Chemistry.

[19]  E. De Clercq,et al.  Synthesis and antiviral activity of novel N-substituted derivatives of acyclovir. , 1988, Journal of medicinal chemistry.

[20]  J. Piškur,et al.  Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster. , 2000, Journal of molecular biology.

[21]  A. Sercel,et al.  Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2′-deoxycytidine for the study of oligonucleotides ☆ , 2004 .

[22]  K. Igarashi,et al.  A deoxythymidine kinase-deficient mutant of Escherichia coli. I. Isolation and some properties. , 1967, Biochimica et biophysica acta.

[23]  J. Knutson,et al.  Fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into oligonucleotides. , 1997, Analytical biochemistry.

[24]  R. Schinazi,et al.  Synthesis and biological evaluation of 2',3'-didehydro-2',3'- dideoxy-5-fluorocytidine (D4FC) analogues: discovery of carbocyclic nucleoside triphosphates with potent inhibitory activity against HIV-1 reverse transcriptase. , 1999, Journal of medicinal chemistry.

[25]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[26]  D. Kristol,et al.  Identification of a pharmacophore for nucleoside analog inhibitors directed at HIV‐1 reverse transcriptase , 1994, Journal of molecular recognition : JMR.

[27]  Mark J. Olsen,et al.  Function-based isolation of novel enzymes from a large library , 2000, Nature Biotechnology.

[28]  J. Piškur,et al.  Four Deoxynucleoside Kinase Activities from Drosophila melanogaster Are Contained within a Single Monomeric Enzyme, a New Multifunctional Deoxynucleoside Kinase* , 1998, The Journal of Biological Chemistry.

[29]  Gerd Folkers,et al.  Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling , 1999, Nature Biotechnology.

[30]  A. Karlsson,et al.  Retroviral Transduction of Cancer Cell Lines with the Gene Encoding Drosophila melanogaster Multisubstrate Deoxyribonucleoside Kinase* , 2000, The Journal of Biological Chemistry.

[31]  J R Barrio,et al.  A Fluorescent Modification of Adenosine Triphosphate with Activity in Enzyme Systems: 1,N6-Ethenoadenosine Triphosphate , 1972, Science.

[32]  J. Piškur,et al.  Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs , 2007, Gene Therapy.

[33]  H. Eklund,et al.  A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines , 2002, The EMBO journal.

[34]  H. Eklund,et al.  Structural basis for feedback inhibition of the deoxyribonucleoside salvage pathway: studies of the Drosophila deoxyribonucleoside kinase. , 2003, Biochemistry.

[35]  C. Galmarini,et al.  Gemcitabine resistance due to deoxycytidine kinase deficiency can be reverted by fruitfly deoxynucleoside kinase, DmdNK, in human uterine sarcoma cells , 2006, Cancer Chemotherapy and Pharmacology.

[36]  K. Johansson,et al.  Structure and function of cellular deoxyribonucleoside kinases , 2002, Cellular and Molecular Life Sciences CMLS.

[37]  S. Chandra,et al.  Synthesis and biological evaluation of 99mTc-DHPM complex: a potential new radiopharmaceutical for lung imaging studies , 2010 .

[38]  S. Lutz,et al.  Non-homologous recombination of deoxyribonucleoside kinases from human and Drosophila melanogaster yields human-like enzymes with novel activities. , 2007, Journal of molecular biology.

[39]  J. Balzarini,et al.  Enhanced toxicity of purine nucleoside analogs in cells expressing Drosophila melanogaster nucleoside kinase mutants , 2007, Gene Therapy.

[40]  F. Moolten.  Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. , 1986, Cancer research.

[41]  Elias S. J. Arnér,et al.  Mammalian deoxyribonucleoside kinases. , 1995, Pharmacology & therapeutics.

[42]  M. Black,et al.  Characterization of Herpes Simplex Virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity , 2002, Protein science : a publication of the Protein Society.

[43]  M. P. Gallagher,et al.  The nucleoside transport proteins, NupC and NupG, from Escherichia coli: specific structural motifs necessary for the binding of ligands. , 2005, Organic & biomolecular chemistry.