title: Characterization of Glycerol Dehydrogenase from Thermoanaerobacterium thermosaccharolyticum DSM 571 and GGG Motif Identification.
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 Mn(2+) enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp(171), His(254), and His(271)) 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 Arg(43), both related to dinucleotide binding.
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Liangliang
authors.initials: L
authors.lastname: Wang
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Jiajun
authors.initials: J
authors.lastname: Wang
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Shi
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Huaxiang
authors.initials: H
authors.lastname: Gu
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Xun
authors.initials: X
authors.lastname: Li
authors.affiliation: College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, P.R. China.|Jiangsu Key Laboratory of Biomass-Based Green Fuels and Chemicals, Nanjing 210037, P.R. China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046950:Thermoanaerobacterium / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D008345:Manganese; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D005990:Glycerol
keywords: Thermoanaerobacterium thermosaccharolyticum; dinucleotide binding; glycerol dehydrogenase; molecular modeling; site-directed mutagenesis