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Biocatalysis and Fermentation Technology
Characterization of Glycerol Dehydrogenase from Thermoanaerobacterium thermosaccharolyticum DSM 571 and GGG Motif Identification
1College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China, 2Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China
Received: December 17, 2015; Accepted: March 9, 2016
J. Microbiol. Biotechnol. 2016; 26(6): 1077-1086
Published June 28, 2016 https://doi.org/10.4014/jmb.1512.12051
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing
its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a
putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571
(TtGlyDH) and expressed in Escherichia coli. The presence of Mn2+ enhanced its enzymatic
activity by 79.5%. Three highly conserved residues (Asp171, His254, and His271) in TtGlyDH were
associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA
reduction, TtGlyDH showed maximum activity towards glycerol at 60°C and pH 8.0 and
towards DHA at 60°C and pH 6.0. DHA reduction was the dominant reaction, with a lower
Km(DHA) of 1.08 ± 0.13 mM and Vmax of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation,
with a Km(glycerol) of 30.29 ± 3.42 mM and Vmax of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent
activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining
65% of its activity after a 2-h incubation at 60°C. Molecular modeling and site-directed
mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif
(GGG motif) and a basic residue Arg43, both related to dinucleotide binding.
Keywords
dinucleotide binding, glycerol dehydrogenase, molecular modeling, site-directed mutagenesis, Thermoanaerobacterium thermosaccharolyticum
References
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
- Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISSMODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.
- Bellamacina CR. 1996. The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 10: 1257-1269.
- Bruins ME, Janssen AE, Boom RM. 2001. Thermozymes and their applications: a review of recent literature and patents. Appl. Biochem. Biotechnol. 90: 155-186.
- Burke J, Ruzheinikov SN, Sedelnikova S, Baker PJ, Holmes D, Muir NM, et al. 2001. Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus. Acta Crystallogr. D Biol. Crystallogr. 57: 165-167.
- Chimtong S, Tachaapaikoon C, Pason P, Kyu KL, Kosugi A, Mori Y, Ratanakhanokchai K. 2011. Isolation and characterization of endocellulase-free multienzyme complex from newly isolated Thermoanaerobacterium thermosaccharolyticum strain NOI-1. J. Microbiol. Biotechnol. 21: 284-292.
- Guex N, Peitsch MC. 1997. SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723.
- Hanukoglu I. 2015. Proteopedia: Rossmann fold: a betaalphabeta fold at dinucleotide binding sites. Biochem. Mol. Biol. Educ. 43: 206-209.
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948.
- Lesk AM. 1995. NAD-binding domains of dehydrogenases. Curr. Opin. Struct. Biol. 5: 775-783.
- Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, et al. 2002. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. USA 99: 11664-11669.
- Mariani V, Kiefer F, Schmidt T, Haas J, Schwede T. 2011. Assessment of template based protein structure predictions in CASP9. Proteins 79 Suppl 10: 37-58.
- Matsuzawa T, Ohashi T, Hosomi A, Tanaka N, Tohda H, Takegawa K. 2010. The gld1+ gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe. Appl. Microbiol. Biotechnol. 87: 715-727.
- Musille P, Ortlund E. 2014. Structure of glycerol dehydrogenase from Serratia. Acta Crystallogr. F Struct. Biol. Commun. 70:166-172.
- Noble JE, Bailey MJ. 2009. Quantitation of protein. Methods Enzymol. 463: 73-95.
- Pei J, Pang Q, Zhao L, Fan S, Shi H. 2012. Thermoanaerobacterium thermosaccharolyticum beta-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose. Biotechnol. Biofuels 5: 31.
- Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
- Raynaud C, Lee J, Sarcabal P, Croux C, Meynial-Salles I, Soucaille P. 2011. Molecular characterization of the glyceroloxidative pathway of Clostridium butyricum VPI 1718. J. Bacteriol. 193: 3127-3134.
- Rosell A, Valencia E, Ochoa WF, Fita I, Pares X, Farres J. 2003. Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase. J. Biol. Chem. 278: 40573-40580.
- Rossmann MG, Moras D, Olsen KW. 1974. Chemical and biological evolution of nucleotide-binding protein. Nature 250: 194-199.
- Ruzheinikov SN, Burke J, Sedelnikova S, Baker PJ, Taylor R, Bullough PA, et al. 2001. Glycerol dehydrogenase: structure, specificity, and mechanism of a family III polyol dehydrogenase. Structure 9: 789-802.
- Scharschmidt M, Pfleiderer G, Metz H, Brummer W. 1983. Isolation and characterization of glycerol dehydrogenase from Bacillus megaterium. Hoppe Seylers Z. Physiol. Chem. 364:911-921.
- Schwede T , Kopp J, G uex N, P eitsch M C. 2 003. S WISSMODEL:an automated protein homology-modeling server. Nucleic Acids Res. 31: 3381-3385.
- Scrutton NS, Berry A, Perham RN. 1990. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343: 38-43.
- Sellek GA, Chaudhuri JB. 1999. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb. Technol. 25: 471-482.
- Spencer P, Bown KJ, Scawen MD, Atkinson T, Gore MG. 1989. Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus. Biochim. Biophys. Acta 994:270-279.
- Truniger V, Boos W. 1994. Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase. J. Bacteriol. 176: 1796-1800.
- Vieille C, Burdette DS, Zeikus JG. 1996. Thermozymes. Biotechnol. Annu. Rev. 2: 1-83.
- Watanabe S, Kodaki T, Makino K. 2005. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280: 10340-10349.
- Wilkinson KW, Baker PJ, Rice DW, Stillman TJ, Gore MG, Krauss O, Atkinson T. 1995. Crystallization of glycerol dehydrogenase from Bacillus stearothermophilus. Acta Crystallogr. D Biol. Crystallogr. 51: 830-832.
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Research article
J. Microbiol. Biotechnol. 2016; 26(6): 1077-1086
Published online June 28, 2016 https://doi.org/10.4014/jmb.1512.12051
Copyright © The Korean Society for Microbiology and Biotechnology.
Characterization of Glycerol Dehydrogenase from Thermoanaerobacterium thermosaccharolyticum DSM 571 and GGG Motif Identification
Liangliang Wang 1, 2, Jiajun Wang 1, 2, Hao Shi 1, 2, Huaxiang Gu 1, 2, Yu Zhang 1, 2, Xun Li 1, 2 and Fei Wang 1, 2*
1College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China, 2Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China
Received: December 17, 2015; Accepted: March 9, 2016
Abstract
Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing
its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a
putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571
(TtGlyDH) and expressed in Escherichia coli. The presence of Mn2+ enhanced its enzymatic
activity by 79.5%. Three highly conserved residues (Asp171, His254, and His271) in TtGlyDH were
associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA
reduction, TtGlyDH showed maximum activity towards glycerol at 60°C and pH 8.0 and
towards DHA at 60°C and pH 6.0. DHA reduction was the dominant reaction, with a lower
Km(DHA) of 1.08 ± 0.13 mM and Vmax of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation,
with a Km(glycerol) of 30.29 ± 3.42 mM and Vmax of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent
activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining
65% of its activity after a 2-h incubation at 60°C. Molecular modeling and site-directed
mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif
(GGG motif) and a basic residue Arg43, both related to dinucleotide binding.
Keywords: dinucleotide binding, glycerol dehydrogenase, molecular modeling, site-directed mutagenesis, Thermoanaerobacterium thermosaccharolyticum
References
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410.
- Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISSMODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.
- Bellamacina CR. 1996. The nicotinamide dinucleotide binding motif: a comparison of nucleotide binding proteins. FASEB J. 10: 1257-1269.
- Bruins ME, Janssen AE, Boom RM. 2001. Thermozymes and their applications: a review of recent literature and patents. Appl. Biochem. Biotechnol. 90: 155-186.
- Burke J, Ruzheinikov SN, Sedelnikova S, Baker PJ, Holmes D, Muir NM, et al. 2001. Purification, crystallization and quaternary structure analysis of a glycerol dehydrogenase S305C mutant from Bacillus stearothermophilus. Acta Crystallogr. D Biol. Crystallogr. 57: 165-167.
- Chimtong S, Tachaapaikoon C, Pason P, Kyu KL, Kosugi A, Mori Y, Ratanakhanokchai K. 2011. Isolation and characterization of endocellulase-free multienzyme complex from newly isolated Thermoanaerobacterium thermosaccharolyticum strain NOI-1. J. Microbiol. Biotechnol. 21: 284-292.
- Guex N, Peitsch MC. 1997. SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723.
- Hanukoglu I. 2015. Proteopedia: Rossmann fold: a betaalphabeta fold at dinucleotide binding sites. Biochem. Mol. Biol. Educ. 43: 206-209.
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948.
- Lesk AM. 1995. NAD-binding domains of dehydrogenases. Curr. Opin. Struct. Biol. 5: 775-783.
- Lesley SA, Kuhn P, Godzik A, Deacon AM, Mathews I, Kreusch A, et al. 2002. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl. Acad. Sci. USA 99: 11664-11669.
- Mariani V, Kiefer F, Schmidt T, Haas J, Schwede T. 2011. Assessment of template based protein structure predictions in CASP9. Proteins 79 Suppl 10: 37-58.
- Matsuzawa T, Ohashi T, Hosomi A, Tanaka N, Tohda H, Takegawa K. 2010. The gld1+ gene encoding glycerol dehydrogenase is required for glycerol metabolism in Schizosaccharomyces pombe. Appl. Microbiol. Biotechnol. 87: 715-727.
- Musille P, Ortlund E. 2014. Structure of glycerol dehydrogenase from Serratia. Acta Crystallogr. F Struct. Biol. Commun. 70:166-172.
- Noble JE, Bailey MJ. 2009. Quantitation of protein. Methods Enzymol. 463: 73-95.
- Pei J, Pang Q, Zhao L, Fan S, Shi H. 2012. Thermoanaerobacterium thermosaccharolyticum beta-glucosidase: a glucose-tolerant enzyme with high specific activity for cellobiose. Biotechnol. Biofuels 5: 31.
- Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
- Raynaud C, Lee J, Sarcabal P, Croux C, Meynial-Salles I, Soucaille P. 2011. Molecular characterization of the glyceroloxidative pathway of Clostridium butyricum VPI 1718. J. Bacteriol. 193: 3127-3134.
- Rosell A, Valencia E, Ochoa WF, Fita I, Pares X, Farres J. 2003. Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase. J. Biol. Chem. 278: 40573-40580.
- Rossmann MG, Moras D, Olsen KW. 1974. Chemical and biological evolution of nucleotide-binding protein. Nature 250: 194-199.
- Ruzheinikov SN, Burke J, Sedelnikova S, Baker PJ, Taylor R, Bullough PA, et al. 2001. Glycerol dehydrogenase: structure, specificity, and mechanism of a family III polyol dehydrogenase. Structure 9: 789-802.
- Scharschmidt M, Pfleiderer G, Metz H, Brummer W. 1983. Isolation and characterization of glycerol dehydrogenase from Bacillus megaterium. Hoppe Seylers Z. Physiol. Chem. 364:911-921.
- Schwede T , Kopp J, G uex N, P eitsch M C. 2 003. S WISSMODEL:an automated protein homology-modeling server. Nucleic Acids Res. 31: 3381-3385.
- Scrutton NS, Berry A, Perham RN. 1990. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343: 38-43.
- Sellek GA, Chaudhuri JB. 1999. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb. Technol. 25: 471-482.
- Spencer P, Bown KJ, Scawen MD, Atkinson T, Gore MG. 1989. Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus. Biochim. Biophys. Acta 994:270-279.
- Truniger V, Boos W. 1994. Mapping and cloning of gldA, the structural gene of the Escherichia coli glycerol dehydrogenase. J. Bacteriol. 176: 1796-1800.
- Vieille C, Burdette DS, Zeikus JG. 1996. Thermozymes. Biotechnol. Annu. Rev. 2: 1-83.
- Watanabe S, Kodaki T, Makino K. 2005. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc. J. Biol. Chem. 280: 10340-10349.
- Wilkinson KW, Baker PJ, Rice DW, Stillman TJ, Gore MG, Krauss O, Atkinson T. 1995. Crystallization of glycerol dehydrogenase from Bacillus stearothermophilus. Acta Crystallogr. D Biol. Crystallogr. 51: 830-832.
