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Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase

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Abstract

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD+ than with NADP+. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C.

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Acknowledgment

This work was supported by the National Research Initiative of the United States Department of Agriculture’s Cooperative State Research, Education, and Extension Service, under grant number 2005-35504-16239. S. H. Song was supported in part by a grant from the Korea Research Foundation, Korean Government (MOEHRD; KRF-2006-214-D00050). L.T.J.D. is a Canada Research Chair in Structural Biochemistry. We thank Dr. J. G. Zeikus for his enthusiastic support and valuable discussions. We are grateful for the use of Dr. Jennifer Ekstrom’s chromatography system. We thank Dr. Joe Leykam and Dr. William Wedemeyer for their assistance with analytical ultracentrifugation and analytical ultracentrifugation data analysis, respectively. We thank Dr. R. M. Kelly from North Carolina State University for sending us a preculture of T. maritima.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michigan State University, 110 Biochemistry Building, East Lansing, MI, 48824, USA

    Seung Hoon Song, Nitasha Ahluwalia & Claire Vieille

  2. Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E5, Canada

    Yvonne Leduc & Louis T. J. Delbaere

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  1. Seung Hoon Song
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  2. Nitasha Ahluwalia
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  5. Claire Vieille
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Correspondence to Claire Vieille.

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Song, S.H., Ahluwalia, N., Leduc, Y. et al. Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase. Appl Microbiol Biotechnol 81, 485–495 (2008). https://doi.org/10.1007/s00253-008-1633-9

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  • DOI: https://doi.org/10.1007/s00253-008-1633-9

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