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Glutamate kinase from Thermotoga maritima: characterization of a thermophilic enzyme for proline biosynthesis

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Abstract

Glutamate kinase (GK), an enzyme involved in osmoprotection in plants and microorganisms, catalyses the first and controlling step of proline biosynthesis. The proB gene encoding GK was cloned from the hyperthermophilic bacterium Thermotoga maritima and overexpressed in Escherichia coli, and the resulting protein was purified to homogeneity in three simple steps. T. maritima GK behaved as a tetramer, showing maximal activity at 83°C, and was inhibited by ADP and proline. Although T. maritima GK exhibited high amino acid similarity to the mesophilic E. coli GK, it was less dependent of Mg ions and was not aggregated in the presence of proline. Moreover, it displayed a greater thermostability and higher catalytic efficiency than its mesophilic counterpart at elevated temperatures.

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Abbreviations

tmGK:

Thermotoga maritima glutamate kinase

ecGK:

Escherichia coli glutamate kinase

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Acknowledgments

We would like to thank Dr. Vicente Rubio for kindly providing the cDNA of T. maritima and the plasmid pGroESL, and Ana Isabel Martínez and Jesús Rodríguez for their helpful insight. The proteomic molecular weight fingerprinting by MS-MALDI-TOF analysis was carried out in the Centro de Investigación Príncipe Felipe, which is a member of the ProteoRed network. This work was financed by Grants BFU 2007/66781 and CIBERER739.

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Authors and Affiliations

  1. Centro de Investigación Príncipe Felipe (CIPF), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII), Avda. El Saler, 16, 46012, Valencia, Spain

    Isabel Pérez-Arellano & Javier Cervera

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  1. Isabel Pérez-Arellano
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  2. Javier Cervera
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Correspondence to Javier Cervera.

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Communicated by F. Robb.

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Pérez-Arellano, I., Cervera, J. Glutamate kinase from Thermotoga maritima: characterization of a thermophilic enzyme for proline biosynthesis. Extremophiles 14, 409–415 (2010). https://doi.org/10.1007/s00792-010-0320-9

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  • DOI: https://doi.org/10.1007/s00792-010-0320-9

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