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Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase

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Abstract

The genes for trehalose synthesis in Thermus thermophilus RQ-1, namely otsA [trehalose-phosphate synthase (TPS)], otsB [trehalose-phosphate phosphatase (TPP)], and treS [trehalose synthase (maltose converting) (TreS)] genes are structurally linked. The TPS/TPP pathway plays a role in osmoadaptation, since mutants unable to synthesize trehalose via this pathway were less osmotolerant, in trehalose-deprived medium, than the wild-type strain. The otsA and otsB genes have now been individually cloned and overexpressed in Escherichia coli and the corresponding recombinant enzymes purified. The apparent molecular masses of TPS and TPP were 52 and 26 kDa, respectively. The recombinant TPS utilized UDP-glucose, TDP-glucose, ADP-glucose, or GDP-glucose, in this order as glucosyl donors, and glucose-6-phosphate as the glucosyl acceptor to produce trehalose-6-phosphate (T6P). The recombinant TPP catalyzed the dephosphorylation of T6P to trehalose. This enzyme also dephosphorylated G6P, and this activity was enhanced by NDP-glucose. TPS had an optimal activity at about 98°C and pH near 6.0; TPP had a maximal activity near 70°C and at pH 7.0. The enzymes were extremely thermostable: at 100°C, TPS had a half-life of 31 min, and TPP had a half-life of 40 min. The enzymes did not require the presence of divalent cations for activity; however, the presence of Co2+ and Mg2+ stimulates both TPS and TPP. This is the first report of the characterization of TPS and TPP from a thermophilic organism.

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Acknowledgements

This research was funded by the European Commission 5th Framework Programme, project QLK3-CT-2000-00640, and FCT/FEDER projects PRAXIS/P/BIO/12082/1998 and POCTI/35715/BIO/2000. Z. Silva acknowledges a Ph.D. grant from PRAXIS XXI (BD/21669/99).

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  1. Departamento de Bioquímica and Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-517, Coimbra, Portugal

    Zélia Silva, Susana Alarico & Milton S. da Costa

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  1. Zélia Silva
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  2. Susana Alarico
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  3. Milton S. da Costa
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Correspondence to Milton S. da Costa.

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Communicated by G. Antranikian

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Silva, Z., Alarico, S. & da Costa, M.S. Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Extremophiles 9, 29–36 (2005). https://doi.org/10.1007/s00792-004-0421-4

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