title: A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System.
abstract: Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic "alphabet". Specifically, we show that DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'β-d-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-β-d-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.|Department of Chemistry, University of Florida , Gainesville, Florida 32611, United States.
authors.forename: Mariko F
authors.identifier: 0000-0002-2072-8141
authors.initials: MF
authors.lastname: Matsuura
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Christian B
authors.initials: CB
authors.lastname: Winiger
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences, LLC , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Shaw
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences, LLC , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Myong-Jung
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences, LLC , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Myong-Sang
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Emory University , 1515 Dickey Drive, Atlanta, Georgia 30322, United States.
authors.forename: Ashley B
authors.initials: AB
authors.lastname: Daugherty
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Fei
authors.initials: F
authors.lastname: Chen
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences, LLC , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Patricia
authors.initials: P
authors.lastname: Moussatche
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Moses
authors.affiliation: Department of Chemistry, Emory University , 1515 Dickey Drive, Atlanta, Georgia 30322, United States.
authors.forename: Stefan
authors.initials: S
authors.lastname: Lutz
authors.affiliation: The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences, LLC , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
authors.forename: Steven A
authors.identifier: 0000-0002-3318-9917
authors.initials: SA
authors.lastname: Benner
mesh_terms: D000596:Amino Acids / Q000235:genetics; D000818:Animals; D020029:Base Pairing / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D006860:Hydrogen Bonding; D009705:Nucleosides / Q000235:genetics*; D010766:Phosphorylation / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011122:Polyphosphates / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D014227:Triazines / Q000378:metabolism
chemical_list: D000596:Amino Acids; D009705:Nucleosides; D011122:Polyphosphates; D011743:Pyrimidines; D014227:Triazines; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; C030986:pyrimidine; C005692:triphosphoric acid
keywords: artificial metabolism; artificially expanded genetic information system; enzyme engineering; nucleoside/nucleotide analogues; nucleotide biosynthesis