Part:BBa_K5392004
ZaTdT-mutant type (ZaTdT-R335F)
Description
To make the active center more compatible with 3’-O-(2-nitrobenzyl)-modified nucleotides, we attempted to reshape the catalytic cavity using mutagenesis on the residues within 6 Å around 3’-O-(2-nitrobenzyl) and the base moiety. We predicted the 3D structure of ZaTdT by homology modelling and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP. The results of molecular docking indicate that residues Arg335 and Lys337 are closely related to the catalytic activity of ZaTdT.
Analysis of the molecular dynamics (MD) trajectories showed that R335 and its spatially neighboring residue K337 in ZaTdT contacted the triphosphate group through hydrogen bonds. We anticipate that eliminating the hydrogen bonds binding force between the above residues and the triphosphate group will release the greater freedom of the modified nucleoside within the active pocket, thereby increasing catalytic activity. Consequently, We performed molecular dynamics simulations of the 3’-O-(2-nitrobenzyl)-dATP docked into ZaTdT and ZaTdT-R335F. The predicted results show that above substitution can abolish the hydrogen-bond connection at position 335.
Experiment
Construction of single-site saturation mutant plasmid
To obtain the desired saturation mutagenesis, oligonucleotide primers were designed with degenerate codons. In addition, each single-site saturation mutant was generated according to the PCR-based QuickChange method. The PCR was performed according to the operation manual. The PCR product was digested with DpnI restriction enzyme and transformed into E.coli DH5-alpha competent cells.
SDS-PAGE of ZaTdT-R335F
We transfected the Sequencing is correct ZaTdT-R335F plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity
Catalytic activity assay of ZaTdT-R335F
We transfected the Sequencing is correct ZaTdT-R335F plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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