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A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H).
FEBS J. 2015 Jun; 282(12):2339-51.FJ

Abstract

Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181-Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes.

Authors+Show Affiliations

State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.Department of Biological Sciences, National University of Singapore, Singapore.State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, China.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

25817922

Citation

Cui, Dongbing, et al. "A Computational Strategy for Altering an Enzyme in Its Cofactor Preference to NAD(H) And/or NADP(H)." The FEBS Journal, vol. 282, no. 12, 2015, pp. 2339-51.
Cui D, Zhang L, Jiang S, et al. A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H). FEBS J. 2015;282(12):2339-51.
Cui, D., Zhang, L., Jiang, S., Yao, Z., Gao, B., Lin, J., Yuan, Y. A., & Wei, D. (2015). A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H). The FEBS Journal, 282(12), 2339-51. https://doi.org/10.1111/febs.13282
Cui D, et al. A Computational Strategy for Altering an Enzyme in Its Cofactor Preference to NAD(H) And/or NADP(H). FEBS J. 2015;282(12):2339-51. PubMed PMID: 25817922.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A computational strategy for altering an enzyme in its cofactor preference to NAD(H) and/or NADP(H). AU - Cui,Dongbing, AU - Zhang,Lujia, AU - Jiang,Shuiqin, AU - Yao,Zhiqiang, AU - Gao,Bei, AU - Lin,Jinping, AU - Yuan,Y Adam, AU - Wei,Dongzhi, Y1 - 2015/04/22/ PY - 2013/07/17/received PY - 2015/03/09/revised PY - 2015/03/23/accepted PY - 2015/3/31/entrez PY - 2015/3/31/pubmed PY - 2015/8/26/medline KW - altered coenzyme specificity KW - molecular dynamics simulation KW - rational computational design KW - site-directed mutagenesis KW - structure stability prediction SP - 2339 EP - 51 JF - The FEBS journal JO - FEBS J. VL - 282 IS - 12 N2 - Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181-Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes. SN - 1742-4658 UR - https://www.unboundmedicine.com/medline/citation/25817922/A_computational_strategy_for_altering_an_enzyme_in_its_cofactor_preference_to_NAD_H__and/or_NADP_H__ L2 - https://doi.org/10.1111/febs.13282 DB - PRIME DP - Unbound Medicine ER -