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Cited by 1Protein Structure and DynamicsAdvanced NMR Techniques and ApplicationsEnzyme Catalysis and Immobilization

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NMR-guided rational exploration of co-factors in boosting the Pfu DNA polymerase

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Chen, Yihao, Zhu, Mingjun, Ding, Ruishen, Zhao, Xiaoling, Tao, Zhiqing, Zhang, Xu, Liu, Maili, He, Lichun·2025·Magnetic Resonance Letters
DOI 10.1016/j.mrl.2025.200218PMC PMC12640072

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Abstract

ABSTRACT With rapid developments of emerging technologies like synthetic biology, the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly. Thus, rational optimization of the performance of DNA polymerase is of great interest. Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique used for studying protein structure and dynamics. It provides the atomic resolution information of enzymes under its functional solution environment to reveal the active sites (hot spots) of the enzyme, which could be further used for optimizing the performance of enzymes. In our previous work, we identified hot spot residues of Pfu DNA polymerase ( Pfu pol). We aim to employ these binding hot spots to screen for co-factors of Pfu pol, particularly targeting those molecules exhibiting weak intermolecular interactions. To validate this concept, we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system. Employing these hot spots as probes, two new co-factors, the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine, are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol. This NMR-based approach requires no prior assignment information of target enzymes, guiding the rational exploration of novel co-factors for Pfu pol. Moreover, our approach is not dependent on structural data or bioinformatics. Therefore, it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.

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Metadata

Authors
Chen, Y., Zhu, M., Ding, R., Zhao, X., Tao, Z., Zhang, X., Liu, M., & He, L.
Affiliations
  • University of Chinese Academy of Sciences (CN)
  • Chinese People's Liberation Army (CN)
Corresponding author
He, Lichun
ORCID
Venue
Magnetic Resonance Letters
Publisher
Elsevier BV
Published
2025-11
Volume
5
Issue
4
Pages
200218
ISSN
  • 2097-0048 (ISSN-L)
  • 2772-5162
DOI
10.1016/j.mrl.2025.200218
PMC
PMC12640072
OpenAlex
W4410593740
Cited by
1
References
1
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Magnetic Resonance Letters (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Chinese Academy of Sciences (XDB0540000)
  • Natural Science Foundation of Hubei Province (2023AFA041)
  • National Natural Science Foundation of China (22174151, 22327901, 21991080)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
Pfu DNA polymerase, Pyrococcus furiosus, cofactors, hot spot residues, protein-protein interaction, thermal stability, polymerase processivity, Thermococcus kodakarensis, L-arginine, chemical chaperones, Tween 20, NMR-based screening, enzyme engineering

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