Loop-Centric Engineering of Terminal Deoxynucleotidyl Transferase for Enhanced Catalysis of 3'-Modified Nucleotides.

IF 3.9 2区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biotechnology and Bioengineering Pub Date : 2026-09-30 DOI:10.1002/bit.70397

Kun Shi, An-Na Li, Jian-He Xu, Hui-Lei Yu

引用次数: 0

Abstract

Efficient enzymatic DNA synthesis requires terminal deoxynucleotidyl transferases (TdTs) that can accept reversibly blocked nucleotides, but improving wild-type enzymes has traditionally depended on labor-intensive screening. Here, we combine deep learning-based structure prediction with molecular dynamics simulations to guide loop-centric engineering of a TdT from Crocodylus porosus. The resulting variant M3 (V253E/L256M/N338R) showed a 26-fold increase in specific activity toward 3'-ONH₂-dCTP while maintaining expression and thermostability. Simulations indicated that the mutations reorganized Loop1 dynamics and reproducibly biased the incoming nucleotide toward a more favorable in-line attack orientation. The key N338R change also improved a second TdT scaffold, supporting N338 as a potentially transferable engineering hotspot. These results provide a practical computational strategy for improving terminal deoxynucleotidyl transferases for incorporation of 3'-modified nucleotides, reducing reliance on large-scale screening.

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末端脱氧核苷酸转移酶的环中心工程增强3'-修饰核苷酸的催化作用。

高效的酶促DNA合成需要能够接受可逆阻断核苷酸的末端脱氧核苷酸转移酶（tdt），但改进野生型酶传统上依赖于劳动密集型筛选。在这里，我们将基于深度学习的结构预测与分子动力学模拟相结合，以指导鳄鱼TdT的环中心工程。得到的变异M3 （V253E/L256M/N338R）在保持表达和热稳定性的同时，对3'-ONH2-dCTP的特异性活性增加了26倍。模拟表明，突变重组了Loop1动力学，并可重复地将进入的核苷酸偏向于更有利的在线攻击方向。关键的N338R变化也改善了第二个TdT支架，支持N338作为潜在的可转移工程热点。这些结果为改善末端脱氧核苷酸转移酶以整合3'-修饰核苷酸提供了实用的计算策略，减少了对大规模筛选的依赖。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Biotechnology and Bioengineering 工程技术-生物工程与应用微生物

CiteScore

7.90

自引率

5.30%

发文量

280

审稿时长

2.1 months

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