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Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli

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Abstract

Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.

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Acknowledgments

The authors LC, PZ, and JS gratefully thank the financial support from the 973 key basic research program with the project 2011CBA00804 and the Chinese Academy of Sciences with the project KSCX2-EW-G-14-1. ZC and APZ were supported by the German Research Foundation (DFG) through the project ZE 542/6-1 and the Hamburg Excellence Initiative project SynBio.

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Authors and Affiliations

  1. Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People’s Republic of China

    Lin Chen

  2. Joint-lab of Systems Biotechnology, IBB/TUHH and TIB/CAS, Tianjin, 300308, People’s Republic of China

    Lin Chen, Zhen Chen, Ping Zheng, Jibin Sun & An-Ping Zeng

  3. Institute for Bioprocess and Biosystems Engineering, Hamburg University of Technology (IBB/TUHH), Denickestrasse 15, 21073, Hamburg, Germany

    Zhen Chen & An-Ping Zeng

  4. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, People’s Republic of China

    Ping Zheng & Jibin Sun

  5. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (TIB/CAS), Tianjin, 300308, People’s Republic of China

    Ping Zheng & Jibin Sun

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  1. Lin Chen
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  2. Zhen Chen
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Correspondence to Jibin Sun or An-Ping Zeng.

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Lin Chen and Zhen Chen equally contributed to this work.

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Chen, L., Chen, Z., Zheng, P. et al. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli . Appl Microbiol Biotechnol 97, 2939–2949 (2013). https://doi.org/10.1007/s00253-012-4176-z

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