计算机建模研究dNTP选择性的分子基础在人类终端deoxynucleotidyltransferase
Egor O Ukladov1,2, Timofey E Tyugashev1, Nikita A Kuznetsov1,2
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia.
Biomolecules
|August 29, 2024
概括
研究人员修改了人类终端脱核样转移酶 (TdT),以克服其dNTP选择性限制. 突变D395N和D395N+E456N显示出均等的选择性,使得生物技术应用更广泛.
科学领域:
- 生物化学 生化学
- 分子生物学分子生物学
- 酶学 是一种酶学.
背景情况:
- 人类终端脱核样转移酶 (TdT) 是V(D) J重组和DNA修复的关键.
- TdT的模板独立DNA合成对寡核酸合成有价值.
- 一个主要的限制是TdT对脱氧核酸三酸盐 (dNTP) 的强烈选择性:dGTP > dTTP ≈ dATP > dCTP.
研究的目的:
- 使用分子动力学研究氨基酸替代对TdT的dNTP选择性的影响.
- 为了确定特定的残留物和相互作用,决定TdT的dNTP偏好.
- 为生物技术用途设计具有改进和均等dNTP选择性的TdT变体.
主要方法:
- 用分子动力学模拟来建模TdT酶的行为.
- 分析的重点是基和氨基酸残留物在395和456.6位之间的相互作用.
- 系统地引入和模拟单双氨基酸替代.
主要成果:
- 基和残留物395/456之间的稳定键对于dNTP的选择性至关重要.
- 两种TdT突变D395N和D395N+E456N在各种dNTP中表现出显著均衡的选择性.
- 与野生类型酶相比,这些突变体表现出较低的偏好偏差.
结论:
- 在位置395和456的氨基酸替代可以有效调节TdT的dNTP选择性.
- 鉴定到的突变为合理的酶设计提供了一条途径.
- 具有均等的dNTP选择性的工程TdT变体对先进的生物技术应用具有前途.
相关概念视频
01:19Allosteric Proteins-ATCase
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
01:27Pharmacogenetics of Phase II Enzymes: N-acetyltransferase, Thiopurine S-methyltransferase, UDP-glucuronosyltransferase
Phase II biotransformation reactions are essential for detoxifying and eliminating xenobiotics, including many pharmaceutical compounds. These reactions typically involve conjugation, the covalent attachment of polar endogenous groups such as glucuronic acid, sulfate, methyl, or acetyl moieties to functional groups introduced during Phase I metabolism. The resulting conjugates are more water-soluble, enabling efficient renal or biliary excretion.The major classes of Phase II enzymes include...
01:28Biosynthesis of Nucleic Acids
Nucleic acid biosynthesis is a fundamental biochemical process that produces the purine and pyrimidine nucleotides essential for DNA and RNA synthesis. This pathway maintains a balanced nucleotide pool, preventing imbalances that could jeopardize genetic integrity and cellular function. Given the crucial role of nucleotides, their synthesis is tightly regulated to ensure proper cellular homeostasis.Purine BiosynthesisThe biosynthesis of purine nucleotides begins with ribose-5-phosphate, a...