Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant.
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.
CAS Key Laboratory of Genome Sciences & Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.|University of Chinese Academy of Sciences, Beijing, China.|Foundation for Applied Molecular Evolution (FfAME), Alachua, Florida, United States of America.
Fei
F
Chen
College of Chemistry, Beijing Normal University, Beijing, China.
Yuan
Y
Zhang
Department of Chemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America.
Ashley B
AB
Daugherty
Foundation for Applied Molecular Evolution (FfAME), Alachua, Florida, United States of America.
Zunyi
Z
Yang
Foundation for Applied Molecular Evolution (FfAME), Alachua, Florida, United States of America.
Ryan
R
Shaw
CAS Key Laboratory of Genome Sciences & Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.|University of Chinese Academy of Sciences, Beijing, China.
Mengxing
M
Dong
Department of Chemistry, Emory University School of Medicine, Atlanta, Georgia, United States of America.
Stefan
S
Lutz
Foundation for Applied Molecular Evolution (FfAME), Alachua, Florida, United States of America.
Steven A
SA
Benner
D000818:Animals; D052157:Aptamers, Nucleotide / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D009005:Monosaccharides / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
C547309:6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone; D052157:Aptamers, Nucleotide; D009005:Monosaccharides; D009711:Nucleotides; D006151:Guanosine; C035861:2-amino-8-(beta-ribofuranosyl)imidazo(1,2-a)-1,3,5-triazin-4-one; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases