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Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis

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Abstract

Two putative glutamate dehydrogenase (GDH) genes (pcal_1031 and pcal_1606) were found in a sulfur-dependent hyperthermophilic archaeon, Pyrobaculum calidifontis. The two genes were then expressed in Escherichia coli, and both of the recombinant gene products showed GDH activity. The two enzymes were then purified to homogeneity and characterized in detail. Although both purified GDHs had a hexameric structure and neither exhibited allosteric regulation, they showed different coenzyme specificities: one was specific for NAD+, the other for NADP+ and different heat activation mechanisms. In addition, there was little difference in the kinetic constants, optimal temperature, thermal stability, optimal pH and pH stability between the two enzymes. The overall sequence identity between the two proteins was very high (81 %), but was not high in the region recognizing the 2′ position of the adenine ribose moiety, which is responsible for coenzyme specificity. This is the first report on the identification of two GDHs with different coenzyme specificities from a single hyperthermophilic archaeon and the definition of their basic in vitro properties.

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Abbreviations

GDH:

l-Glutamate dehydrogenase

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Acknowledgments

This work was supported in part by Grant-in-Aid for Scientific Research 22248010 (to T.O.) from the Ministry of Education, Science, Sports and Culture of Japan.

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Author notes

  1. Taketo Ohmori

    Present address: Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan

Authors and Affiliations

  1. Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki Higashi-ku, Fukuoka, 812-8581, Japan

    Taisuke Wakamatsu, Chisato Higashi, Taketo Ohmori, Katsumi Doi & Toshihisa Ohshima

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  1. Taisuke Wakamatsu
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  2. Chisato Higashi
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  3. Taketo Ohmori
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  4. Katsumi Doi
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  5. Toshihisa Ohshima
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Correspondence to Taisuke Wakamatsu.

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Communicated by F. Robb.

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Wakamatsu, T., Higashi, C., Ohmori, T. et al. Biochemical characterization of two glutamate dehydrogenases with different cofactor specificities from a hyperthermophilic archaeon Pyrobaculum calidifontis . Extremophiles 17, 379–389 (2013). https://doi.org/10.1007/s00792-013-0527-7

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