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Removal of Feedback Inhibition of Corynebacterium glutamicum Phosphoenolpyruvate Carboxylase by Addition of a Short Terminal Peptide

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Abstract

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the carboxylation of phosphoenolpyruvate (PEP) in the presence of bicarbonate to form oxaloacetate (OAA), and it plays an important role in high-efficient production of OAA-derived metabolites such as lysine, glutamate and succinate. However, PEPCs often suffered from serious feedback inhibition by various metabolic effectors like aspartate. Here, the feedback inhibition of PEPC from Corynebacterium glutamicum was removed by adding a short terminal peptide like His-tag. The effect of His-tag location on the structure and important properties such as activity, thermostability and feedback inhibition of PEPC has been investigated. The purified untagged PEPC, Nterminal His-tagged PEPC (PEPC-N-His) and C-terminal His-tagged PEPC (PEPC-C-His) were characterized. PEPCN- His (439.71/sec/mM) showed a 1.26 and 186-fold higher catalytic efficiency than untagged PEPC (348.59/sec/mM) and PEPC-C-His (2.36/sec/mM), respectively. Both PEPCN- His and untagged PEPC were significantly inhibited by aspartate at the concentrations above 4 mM (residual activities < 10%), while PEPC-C-His was almost desensitized to aspartate within 10 mM (around 90% of residual activity). Structural analysis showed that the extension of C-terminus may cause steric hindrance for aspartate binding with enzymes, leading to the deregulation of feedback inhibition of PEPC-C-His. This study provides a deeper understanding of the effect of terminal fragments on the structure and function of PEPCs, and helps to engineer the feedback inhibition of PEPCs and structurally similar enzymes.

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  1. These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300-457, China

    Deyu Xu & Shuxin Zhao

  2. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300-308, China

    Deyu Xu, Jing Zhao, Guoqiang Cao, Jinyu Wang, Qinggang Li, Ping Zheng & Jibin Sun

  3. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300-308, China

    Jing Zhao, Guoqiang Cao, Jinyu Wang, Qinggang Li, Ping Zheng & Jibin Sun

Authors
  1. Deyu Xu
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  2. Jing Zhao
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  3. Guoqiang Cao
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  4. Jinyu Wang
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  5. Qinggang Li
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  7. Shuxin Zhao
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Correspondence to Ping Zheng.

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Xu, D., Zhao, J., Cao, G. et al. Removal of Feedback Inhibition of Corynebacterium glutamicum Phosphoenolpyruvate Carboxylase by Addition of a Short Terminal Peptide. Biotechnol Bioproc E 23, 72–78 (2018). https://doi.org/10.1007/s12257-017-0313-y

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  • DOI: https://doi.org/10.1007/s12257-017-0313-y

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