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 p...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2017-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5360312?pdf=render |
| _version_ | 1830492562619629568 |
|---|---|
| author | Fei Chen Yuan Zhang Ashley B Daugherty Zunyi Yang Ryan Shaw Mengxing Dong Stefan Lutz Steven A Benner |
| author_facet | Fei Chen Yuan Zhang Ashley B Daugherty Zunyi Yang Ryan Shaw Mengxing Dong Stefan Lutz Steven A Benner |
| author_sort | Fei Chen |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T20:31:05Z |
| format | Article |
| id | doaj.art-fe74bec01f5a4997b06753a97f93b3a1 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T20:31:05Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-fe74bec01f5a4997b06753a97f93b3a12022-12-21T18:51:13ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01123e017416310.1371/journal.pone.0174163Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant.Fei ChenYuan ZhangAshley B DaughertyZunyi YangRyan ShawMengxing DongStefan LutzSteven A BennerOne 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.http://europepmc.org/articles/PMC5360312?pdf=render |
| spellingShingle | Fei Chen Yuan Zhang Ashley B Daugherty Zunyi Yang Ryan Shaw Mengxing Dong Stefan Lutz Steven A Benner Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant. PLoS ONE |
| title | Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant. |
| title_full | Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant. |
| title_fullStr | Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant. |
| title_full_unstemmed | Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant. |
| title_short | Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant. |
| title_sort | biological phosphorylation of an unnatural base pair ubp using a drosophila melanogaster deoxynucleoside kinase dmdnk mutant |
| url | http://europepmc.org/articles/PMC5360312?pdf=render |
| work_keys_str_mv | AT feichen biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT yuanzhang biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT ashleybdaugherty biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT zunyiyang biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT ryanshaw biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT mengxingdong biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT stefanlutz biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant AT stevenabenner biologicalphosphorylationofanunnaturalbasepairubpusingadrosophilamelanogasterdeoxynucleosidekinasedmdnkmutant |