Abstract
Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathionedependent formaldehyde dehydrogenase.
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Abbreviations
- ADH:
-
alcohol dehydrogenase
- CD:
-
circular dichroism
- GSH:
-
glutathione
- GSH-FDH:
-
glutathione-dependent formaldehyde dehydrogenase
References
De Pouplana, L. R., Atrian, S., Gonzalex-Duarte, R., Fothergill-Gilmore, L. A., Kelly, S. M., and Price, N. C. (1991) Structural properties of long-and short-chain alcohol dehydrogenases. Contribution of NAD+ to stability, Biochem. J., 276, 433–438.
Burdette, D. S., Jung, S. H., Shen, G. J., Hollingsworth, R. I., and Zeikus, J. G. (2002) Physiological function of alcohol dehydrogenases and long-chain (C30) fatty acids in alcohol tolerance of Thermoanaerobacter ethanolicus, Appl. Environ. Microbiol., 68, 1914–1918.
Reid, M. F., and Fewson, C. A. (1994) Molecular characterization of microbial alcohol dehydrogenases, Crit. Rev. Microbiol., 20, 13–56.
Machielsen, R., Uria, R. A., Kengen, W. M., and Oost, J. (2006) Production and characterization of a thermostable alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily, Appl. Environ. Microbiol., 72, 233–238.
Kallberg, Y., Oppermann, U., Jornvall, H., and Persson, B. (2002) Short-chain dehydrogenases/reductases (SDRs), Eur. J. Biochem., 269, 4409–4417.
Persson, B., Hedlund, J., and Jornvall, H. (2008) Mediumand short-chain dehydrogenase/reductase gene and protein families, Cell. Mol. Life Sci., 65, 3879–3894.
Danielsson, O., and Jornvall, H. (1992) “Enzymogenesis”: classical liver alcohol dehydrogenase origin from the glutathione-dependent formaldehyde dehydrogenase line, Proc. Natl. Acad. Sci. USA, 89, 9247–9251.
Koivusalo, M., Baumann, M., and Uotila, L. (1989) Evidence for the identity of glutathione-dependent formaldehyde dehydrogenase and class III alcohol dehydrogenase, FEBS Lett., 257, 105–109.
Danielsson, O., Atrian, S., Luque, T., Hjelmqvist, L., Gonzalez-Duarte, R., and Jornvall, H. (1994) Fundamental molecular differences between alcohol dehydrogenase classes, Proc. Natl. Acad. Sci. USA, 91, 4980–4984.
Mashford, P. M., and Jones, A. R. (1982) Formaldehyde metabolism by the rat: a re-appraisal, Xenobiotica, 12, 119–124.
Wagner, F. W., Pares, X., Holmquist, B., and Vallee, B. L. (1984) Physical and enzymatic properties of a class III isozyme of human liver alcohol dehydrogenase: chi-ADH, Biochemistry, 23, 2193–2199.
Barber, R. D., Rott, M. A., and Donohue, T. J. (1996) Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides, J. Bacteriol., 178, 1386–1393.
Canestro, C., Albalat, R., Hjelmqvist, L., Godoy, L., Jornvall, H., and Gonzalez-Duarte, R. (2002) Ascidian and amphioxus Adh genes correlate functional and molecular features of the ADH family expansion during vertebrate evolution, J. Mol. Evol., 54, 81–89.
Cederlund, E., Hedlund, J., Hjelmqvist, L., Jonsson, A., Shafqat, J., Norin, A., Keung, W. M., Persson, B., and Jornvall, H. (2011) Characterization of new medium-chain alcohol dehydrogenases adds resolution to duplications of the class I/III and the sub-class I genes, Chem. Biol. Interact., 191, 8–13.
Sanghani, P. C., Robinson, H., Bosron, W. F., and Hurley, T. D. (2002) Human glutathione-dependent formaldehyde dehydrogenase, structures of apo, binary, and inhibitory ternary complexes, Biochemistry, 41, 10778–10786.
Eklund, H., Plapp, B. V., Samama, J. P., and Branden, C. I. (1982) Binding of substrate in a ternary complex of horse liver alcohol dehydrogenase, J. Biol. Chem., 257, 1434914358.
Jalal, A., Rashid, N., Rasool, N., and Akhtar, M. (2009) Gene cloning and characterization of a xylanase from a newly isolated Bacillus subtilis strain R5, J. Biosci. Bioeng., 107, 360–365.
Kato, N., Yamagami, T., Shimo, M., and Sakazawa, C. (1986) Formaldehyde dismutase, a novel NAD-binding oxidoreductase from Pseudomonas putida F61, Eur. J. Biochem., 156, 59–64.
Yamasue, Y., Tanisaka, T., and Kusanagi, T. (1990) Alcohol dehydrogenase zymogram, its inheritance and anaerobic germinability of seeds of Echinochloa weeds, Japan J. Breed., 40, 53–61.
Hollrigl, V., Hollmann, F., Kleeb, A. C., Buehler, K., and Schmid, A. (2008) TADH, the thermostable alcohol dehydrogenase from Thermus sp. ATN1: a versatile new biocatalyst for organic synthesis, Appl. Microbiol. Biotechnol., 81, 263–273.
Nian, H., Meng, Q., Zhang, W., and Chen, L. (2013) Overexpression of the formaldehyde dehydrogenase gene from Brevibacillus brevis to enhance formaldehyde tolerance and detoxification of tobacco, Appl. Biochem. Biotechnol., 169, 170–180.
Liao, Y., Chen, S., Wang, D., Zhang, W., Wang, S., Ding, J., and Zhu, H. (2013) Structure of formaldehyde dehydrogenase from Pseudomonas aeruginosa: the binary complex with the cofactor NAD+, Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 69, 967–972.
Sakamoto, A., Ueda, M., and Morikawa, H. (2002) Arabidopsis glutathione-dependent formaldehyde dehy-drogenase is an S-nitrosoglutathione reductase, FEBS Lett., 515, 20–24.
Bottoms, C. A., Smith, P. E., and Tanner, J. J. (2002) A structurally conserved water molecule in Rossmann dinucleotide-binding domains, Protein Sci., 11, 2125–2137.
Yang, Z. N., Bosron, W. F., and Hurley, T. D. (1997) Structure of human chi chi alcohol dehydrogenase: a glutathione-dependent formaldehyde dehydrogenase, J. Mol. Biol., 265, 330–343.
Vitale, A., Rosso, F., Barbarisi, A., Labella, T., and D’Auria, S. (2010) Properties and evolution of an alcohol dehydrogenase from the crenarchaeota Pyrobaculum aerophilum, Gene, 46, 26–31.
Ryzewski, C. N., and Pietruszko, R. (1977) Horse liver alcohol dehydrogenase SS: purification and characterization of the homogeneous isoenzyme, Arch. Biochem. Biophys., 183, 73–82.
Woronick, C. L. (1975) Alcohol dehydrogenase from human liver, Methods Enzymol., 41, 369–374.
Tanaka, N., Kusakabe, Y., Ito, K., Yoshimoto, T., and Nakamura, K. T. (2003) Crystal structure of glutathioneindependent formaldehyde dehydrogenase, Chem. Biol. Interact., 143, 211–218.
Tanaka, N., Kusakabe, Y., Ito, K., Yoshimoto, T., and Nakamura, K. T. (2002) Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenases, J. Mol. Biol., 324, 519–533.
Man, H., Gargiulo, S., Frank, A., Hollmann, F., and Grogan, G. (2014) Structure of the NADH-dependent thermostable alcohol dehydrogenase TADH from Thermus sp. ATN1 provides a platform for engineering specificity and improved compatibility with inorganic cofactor-regeneration catalysts, J. Mol. Catal. B Enzymol., 105, 1–6.
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Published in Russian in Biokhimiya, 2017, Vol. 82, No. 1, pp. 64-75. Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM16-211, October 31, 2016.
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Ashraf, R., Rashid, N., Basheer, S. et al. Glutathione-dependent formaldehyde dehydrogenase homolog from Bacillus subtilis strain R5 is a propanol-preferring alcohol dehydrogenase. Biochemistry Moscow 82, 13–23 (2017). https://doi.org/10.1134/S0006297917010023
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DOI: https://doi.org/10.1134/S0006297917010023