Abstract
N-acetyl glutamate kinase (NAGK) is a key enzyme in the synthesis of L-arginine, and L-arginine-sensitive NAGK typically has hexameric architecture. Defining the relationship between this architecture and L-arginine inhibition can provide a foundation to identify the key amino acids involved in the allosteric regulation network of L-arginine. In the present study, the key amino acids in the N-terminal helix (N-helix) of Corynebacterium glutamicum (Cg) NAGK required for hexamer formation were determined using structural homology modeling and site-directed mutagenesis. It was also verified that hexameric architecture is required for the positive cooperativity of inhibition by L-arginine and for efficient catalysis, but that it is not the determinant of inhibition by L-arginine. Monomeric mutants retained a similar sensitivity to L-arginine as the hexameric form, indicating that monomers contain an independent, sensitive signal transduction network of L-arginine to mediate allosteric regulation. Mutation studies of CgNAGKs also revealed that amino acid residues 18–23 of the N-helix are required for inhibition by L-arginine, and that E19 may be an essential amino acid influencing the apparent affinity of L-arginine. Collectively, these studies may illuminate the basic mechanism of metabolic homeostasis of C. glutamicum.
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Acknowledgments
The authors are grateful for the financial support of the National Natural Science Foundation of China (No. 31171633) to S Zheng and the National Key Technology Support Program to Y Lin (No. 2013BAD10B01) and the Fundamental Research Funds for the Central Universities to Y Lin. This work was also partially supported by startup funds from the School of Bioscience and Biotechnology, South China University of Technology and the Key Laboratory of Renewable Energy, Chinese Academy of Sciences (No. y507k81001) to X Yang.
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Huang, Y., Li, C., Zhang, H. et al. Monomeric Corynebacterium glutamicum N-acetyl glutamate kinase maintains sensitivity to L-arginine but has a lower intrinsic catalytic activity. Appl Microbiol Biotechnol 100, 1789–1798 (2016). https://doi.org/10.1007/s00253-015-7065-4
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DOI: https://doi.org/10.1007/s00253-015-7065-4