Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase

  • 211 Accesses

  • 17 Citations

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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Aarnikunnas J, Ronnholm K, Palva A (2002) The mannitol dehydrogenase gene (mdh) from Leuconostoc mesenteroides is distinct from other known bacterial mdh genes. Appl Microbiol Biotechnol 59:665–671

  2. Albery W, Barlett P, Cass A, Sim K (1987) Amperometric enzyme electrodes. Part IV. An enzyme electrode for ethanol. J Electroanal Chem 218:127–134

  3. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) (1993) In: Current protocols in molecular biology. Greene Publishing, New York

  4. Banfield MJ, Salvucci ME, Baker EN, Smith CA (2001) Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 A resolution. J Mol Biol 306:239–250

  5. Bardea A, Katz E, Buckmann AF, Willner I (1997) NAD-dependent enzyme electrodes electrical contact of cofactor dependent enzymes and electrodes. J Am Chem Soc 119:9114–9119

  6. Bertini I, Luchinat C (1984) High spin cobalt(II) as a probe for the investigation of metalloproteins. Adv Inorg Biochem 6:71–111

  7. Brünker P, Altenbuchner J, Kulbe KD, Mattes R (1997) Cloning, nucleotide sequence and expression of a mannitol dehydrogenase from Pseudomonas fluorescens DSM 50106 in Escherichia coli. Biochim Biophys Acta 1351:157–167

  8. Carugo O, Argos P (1997) NADP-dependent enzymes. I: conserved stereochemistry of cofactor binding. Proteins 28:10–28

  9. Chen T, Calabrese Barton S, Binyamin G, Gao Z, Zhang Y, Kim HH, Heller A (2001) A miniature biofuel cell. J Am Chem Soc 123:8630–8631

  10. Gründig B, Wittstock G, Rüdel U, Strehlitz B (1995) Mediator-modified electrodes for electrocatalytic oxidation of NADH. J Electroanal Chem 395:143–157

  11. Hahn G, Kaup B, Bringer-Meyer S, Sahm H (2003) A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene. Arch Microbiol 179:101–107

  12. Hunt JB, Neece SH, Schachman HK, Ginsburg A (1984) Mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase. J Biol Chem 259:14793–14803

  13. Jaegfeldt H, Torstensson A, Gorton I, Johansson G (1981) Catalytic oxidation of reduced nicotinamide adenine dinucleotide by graphite electrodes modified with adsorbed aromatic containing catechol functionalities. Anal Chem 53:1979–1982

  14. Jaenicke R (1991) Protein stability and molecular adaptation to extreme conditions. Eur J Biochem 202:715–728

  15. Johnson MR, Conners SB, Montero CI, Chou CJ, Shockley KR, Kelly RM (2006) The Thermotoga maritima phenotype is impacted by syntrophic interaction with Methanococcus jannaschii in hyperthermophilic coculture. Appl Environ Microbiol 72:811–818

  16. Jörnvall H, von Bahr-Lindstrom H, Jeffery J (1984) Extensive variations and basic features in the alcohol dehydrogenase-sorbitol dehydrogenase family. Eur J Biochem 140:17–23

  17. Jörnvall H, Persson B, Jeffery J (1987) Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases. Eur J Biochem 167:195–201

  18. Karyakin AA, Bobrova OA, Karyakina EE (1995) Electroreduction of NAD+ to enzymatically active NADH at poly(neutral red) modified electrodes. J Electroanal Chem 399:179–184

  19. Kaup B, Bringer-Meyer S, Sahm H (2005) d-Mannitol formation from d-glucose in a whole-cell biotransformation with recombinant Escherichia coli. Appl Microbiol Biotechnol 69:397–403

  20. Korakli M, Vogel RF (2003) Purification and characterization of mannitol dehydrogenase from Lactobacillus sanfranciscensis. FEMS Microbiol Lett 220:281–286

  21. Kulbe KD, Schwab U, Gudernatsch W (1987) Enzyme-catalyzed production of mannitol and gluconic acid. Product recovery by various procedures. Ann N Y Acad Sci 506:552–568

  22. Laurinavicius V, Kurtinaitiene B, Gureviciene V, Boguslavsky L, Geng L, Skotheim T (1996) Amperometric glyceride biosensor. Anal Chem Acta 330:159–166

  23. Le AS, Mulderrig KB (2001) Sorbitol and mannitol. In: O’Brien Nabors L (ed) Alternative sweeteners. Marcel Dekker, New York

  24. Magonet E, Hayen P, Delforge D, Delaive E, Remacle J (1992) Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase. Biochem J 287:361–365

  25. Munteanu FD, Kubota LT, Gorton L (2001) Effect of pH on the catalytic electrooxidation of NADH using different two-electron mediators immobilised on zirconium phosphate. J Electroanal Chem 509:2–10

  26. Nordling E, Jörnvall H, Persson B (2002) Medium-chain dehydrogenases/reductases (MDR). Eur J Biochem 269:4267–4276

  27. Ohtani M, Kuwabata S, Yoneyama H (1997) Electrochemical oxidation of reduced nicotinamide coenzymes at Au electrodes modified with phenothiazine derivative monolayers. J Electroanal Chem 422:45–54

  28. Pariente F, Lorenzo E, Tobalina F, Abruna HD (1995) Aldehyde biosensor based on the determination of NADH enzymatically generated by aldehyde dehydrogenase. Anal Chem 67:3936–3944

  29. Park DH, Zeikus JG (2003) Improved fuel cell and electrode designs for producing electricity from microbial degradation. Biotechnol Bioeng 8:348–355

  30. Park DH, Laivenieks M, Guettler MV, Jain MK, Zeikus JG (1999) Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production. Appl Environ Microbiol 65:2912–2917

  31. Park DH, Vieille C, Zeikus JG (2003) Bioelectrocatalysts: engineered oxidoreductase system for utilization of fumarate reductase in chemical synthesis, detection, and fuel cells. Appl Biochem Biotechnol 111:41–53

  32. Prodomidis MI, Karayannis MI (2002) Enzyme based amperometric biosensors for food analysis. Electroanalysis 14:241–261

  33. Puttick P, Vieille C, Song SH, Fodje MN, Grochulski P, Delbaere LTJ (2007) Crystallization, preliminary X-ray diffraction and structure analysis of Thermotoga maritima mannitol dehydrogenase. Acta Crystallogr F 63:350–352

  34. Sasaki Y, Laivenieks M, Zeikus JG (2005) Lactobacillus reuteri ATCC 53608 mdh gene cloning and recombinant mannitol dehydrogenase characterization. Appl Microbiol Biotechnol 68:36–41

  35. Schafer A, Stein MA, Schneider KH, Giffhorn F (1997) Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme. Appl Microbiol Biotechnol 48:47–52

  36. Schneider KH, Giffhorn F (1989) Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides. Eur J Biochem 184:15–19

  37. Schneider KH, Giffhorn F, Kaplan S (1993) Cloning, nucleotide sequence and characterization of the mannitol dehydrogenase gene from Rhodobacter sphaeroides. J Gen Microbiol 139:2475–2484

  38. Slatner M, Nidetzky B, Kulbe KD (1999) Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens. Biochemistry 38:10489–10498

  39. Soetaert W, Buchholz K, Vandamme EJ (1995) Production of d-mannitol and d-lactic acid by fermentation with Leuconostoc mesenteroides. Agro Food Ind Hi Tech 6:41–44

  40. Soetaert W, Vanhooren P, Vandamme E (1999) Production of mannitol by fermentation methods. Biotechnology 10:261–275

  41. Sriprapundh D, Vieille C, Zeikus JG (2003) Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH. Protein Eng 16:683–690

  42. Stoop JM, Mooibroek H (1998) Cloning and characterization of NADP-mannitol dehydrogenase cDNA from the button mushroom, Agaricus bisporus, and its expression in response to NaCl stress. Appl Environ Microbiol 64:4689–4696

  43. Tomazic SJ, Klibanov AM (1988) Mechanisms of irreversible thermal inactivation of Bacillus α-amylases. J Biol Chem 263:3086–3091

  44. Vallee BL, Auld DS (1990) Zinc coordination, function, structure of zinc enzymes and other proteins. Biochemistry 29:5648–5659

  45. Vallee BL, Galdes A (1984) The metallobiochemistry of zinc enzymes. Adv Enzymol Relat Areas Mol Biol 56:283–430

  46. van der Donk WA, Zhao H (2003) Recent developments in pyridine nucleotide regeneration. Curr Opin Biotechnol 14:421–426

  47. von Weymarn N, Kiviharju K, Leisola M (2002) High-level production of d-mannitol with membrane cell-recycle bioreactor. J Ind Microbiol Biotechnol 29:44–49

  48. Willner I, Katz E (2000) Integration of layered redox proteins and conductive supports for bioelectronic applications. Angew Chem Int Ed 39:1180–1218

  49. Woodyer R, van der Donk WA, Zhao H (2003) Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design. Biochemistry 42:11604–11614

  50. Wu JT, Wu LH, Knight JA (1986) Stability of NADPH: effect of various factors on the kinetics of degradation. Clin Chem 32:314–319

Download references

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.

Author information

Correspondence to Claire Vieille.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Keywords

  • Thermotoga maritima
  • Mannitol dehydrogenase
  • Thermostable enzyme
  • Mannitol
  • Glucose
  • Fructose