The Foundation for Applied Molecular Evolution FfAME

Alachua, Florida, United States

The Foundation for Applied Molecular Evolution FfAME

Alachua, Florida, United States

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PubMed | The Foundation for Applied Molecular Evolution FfAME and Emory University
Type: | Journal: ACS synthetic biology | Year: 2016

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--d-2-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1--d-2-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1-d-2-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1--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.

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