title: Modifying human thymidylate kinase to potentiate azidothymidine activation.
abstract: Based on the knowledge of the crystal structures of yeast and Escherichia coli thymidylate kinases (TmpKs) and the observation that TmpK from E. coli can phosphorylate azidothymidine monophosphate (AZT-MP) much more efficiently than either the yeast or the highly homologous human enzyme, we have engineered yeast and human TmpKs to obtain enzymes that have dramatically improved AZT-MP phosphorylation properties. These modified enzymes have properties that make them attractive candidates for gene therapeutic approaches to potentiating the action of AZT as an inhibitor of human immunodeficiency virus (HIV) replication. In particular, insertion of the lid domain of the bacterial TmpK into the human enzyme results in a pronounced change of the acceptance of AZT-MP such that it is now phosphorylated even faster than TMP.
authors.affiliation: Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, D-37070 Göttingen, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Brundiers
authors.forename: A
authors.initials: A
authors.lastname: Lavie
authors.forename: T
authors.initials: T
authors.lastname: Veit
authors.forename: J
authors.initials: J
authors.lastname: Reinstein
authors.forename: I
authors.initials: I
authors.lastname: Schlichting
authors.forename: N
authors.initials: N
authors.lastname: Ostermann
authors.forename: R S
authors.initials: RS
authors.lastname: Goody
authors.forename: M
authors.initials: M
authors.lastname: Konrad
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D054306:Dideoxynucleotides; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000493:pharmacokinetics; D015215:Zidovudine / Q000031:analogs & derivatives / Q000493:pharmacokinetics*
chemical_list: D054306:Dideoxynucleotides; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase