Research output: Contribution to journal › Article › peer-review
Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase. / Ukladov, Egor O.; Tyugashev, Timofey E.; Kuznetsov, Nikita A.
In: Biomolecules, Vol. 14, No. 8, 961, 08.2024.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Computational Modeling Study of the Molecular Basis of dNTP Selectivity in Human Terminal Deoxynucleotidyltransferase
AU - Ukladov, Egor O.
AU - Tyugashev, Timofey E.
AU - Kuznetsov, Nikita A.
N1 - This work was supported by the Ministry of Science and Higher Education of the Russian Federation, agreement No. 075-15-2022-263.
PY - 2024/8
Y1 - 2024/8
N2 - Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme’s selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants—containing either substitution D395N or substitutions D395N+E456N—that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.
AB - Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme’s selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants—containing either substitution D395N or substitutions D395N+E456N—that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.
KW - DNA synthesis
KW - TdT
KW - dNTP
KW - enzyme
KW - molecular dynamics
KW - molecular modeling
KW - polymerase
KW - rational design
KW - terminal deoxynucleotidyl transferase
UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85202464391&origin=inward&txGid=d645cc666fd99efcc0c44a264dea1c1b
UR - https://www.mendeley.com/catalogue/6b07a4d2-99f6-3c3a-87e5-ad378945421d/
U2 - 10.3390/biom14080961
DO - 10.3390/biom14080961
M3 - Article
C2 - 39199349
VL - 14
JO - Biomolecules
JF - Biomolecules
SN - 2218-273X
IS - 8
M1 - 961
ER -
ID: 60829076