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

KW - Humans

KW - Molecular Dynamics Simulation

KW - DNA Nucleotidylexotransferase/metabolism

KW - Substrate Specificity

KW - Deoxyribonucleotides/metabolism

KW - Thymine Nucleotides/metabolism

KW - Deoxycytosine Nucleotides/metabolism

KW - Deoxyadenine Nucleotides/metabolism

KW - Hydrogen Bonding

KW - Deoxyguanine Nucleotides/metabolism

KW - Amino Acid Substitution

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 -