Date Published: March 21, 2017
Publisher: Public Library of Science
Author(s): Fei Chen, Yuan Zhang, Ashley B. Daugherty, Zunyi Yang, Ryan Shaw, Mengxing Dong, Stefan Lutz, Steven A. Benner, Giovanni Maga.
One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1’-b-d-2’-deoxyribofuranosyl)-2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1’-b-d-2’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo.
Genetic alphabet expansion with unnatural base pairs (UBPs) represents an important embranchment of chemical synthetic biology and is a key focus of synthetic biology . Over the past twenty-five years, scientists have made considerable progress in genetic alphabet expansion by synthesizing different types of nucleotide analogs. Thus far, different UBPs designed based on different hydrogen bonding patterns and/or shape complementarities have been successfully developed by Benner [2–5], Hirao [6–9] and Romesberg [9–14]. Among them, three UBPs (Z-P, developed by Benner SA; Ds-Px, developed by Hirao I; and 5SICS-NaM, developed by Romesberg FE) have been particularly successful, and the DNA fragments containing these UBPs can be efficiently and faithfully amplified in vitro by PCR (fidelity >99%) [3, 8, 10].
The phosphorylation activities of DmdNK and DmdNK-Q81E with different deoxyribonucleosides (dC / dG and dZ / dP) were examined under multiple turnover conditions. The kinetic parameters kcat and KM were derived from the Eadie-Hofstee plots (Table 1), and the catalytic efficiency was determined by kcat/KM [24, 26].
The deoxyribonucleoside kinase from Drosophila melanogaster (DmdNK) is known to efficiently phosphorylate all four natural nucleosides and many nucleoside analogs [21–26]. Because of the broad substrate specificity and the high catalytic efficiency, the structure and function of DmdNK have been intensively investigated by many researchers. The formation of many complex crystal structures between DmdNK and its different natural/unnatural substrates or its feedback inhibitors have been reported [29–34]. Additionally, successful DmdNK mutants have been constructed to phosphorylate specific nucleoside analogs [24, 35–38].