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Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea

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Abstract

Phosphoglycerate mutases (PGM) catalyze the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate as part of glycolysis and gluconeogenesis. Two structural and mechanistically unrelated types of PGMs are known, a cofactor (2,3-bisphosphoglycerate)-dependent (dPGM) and a cofactor-independent enzyme (iPGM). Here, we report the characterization of the first archaeal cofactor-dependent PGM from Thermoplasma acidophilum, which is encoded by ORF TA1347. This ORF was cloned and expressed in Escherichia coli and the recombinant protein was characterized as functional dPGM. The enzyme constitutes a 46 kDa homodimeric protein. Enzyme activity required 2,3-bisphosphoglycerate as cofactor and was inhibited by vanadate, a specific inhibitor of dPGMs in bacteria and eukarya; inhibition could be partially relieved by EDTA. Histidine 23 of the archaeal dPGM of T. acidophilum, which corresponds to active site histidine in dPGMs from bacteria and eukarya, was exchanged for alanine by site directed mutagenesis. The H23A mutant was catalytically inactive supporting the essential role of H23 in catalysis of the archaeal dPGM. Further, an archaeal cofactor-independent PGM encoded by ORF AF1751 from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus was characterized after expression in E. coli. The monomeric 46 kDa protein showed cofactor-independent PGM activity and was stimulated by Mn2+ and exhibited high thermostability up to 70°C. A comprehensive phylogenetic analysis of both types of archaeal phosphoglycerate mutases is also presented.

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  1. Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118, Kiel, Germany

    Ulrike Johnsen & Peter Schönheit

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  1. Ulrike Johnsen
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  2. Peter Schönheit
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Correspondence to Peter Schönheit.

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Communicated by G. Antranikian.

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Johnsen, U., Schönheit, P. Characterization of cofactor-dependent and cofactor-independent phosphoglycerate mutases from Archaea. Extremophiles 11, 647–657 (2007). https://doi.org/10.1007/s00792-007-0094-x

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  • DOI: https://doi.org/10.1007/s00792-007-0094-x

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