J. Microbiol. Biotechnol. (2016), 26(6), 1077–1086 http://dx.doi.org/10.4014/jmb.1512.12051 Research Article Review jmb Characterization of Glycerol Dehydrogenase from Thermoanaerobacterium thermosaccharolyticum DSM 571 and GGG Motif Identification Liangliang Wang1,2, Jiajun Wang1,2, Hao Shi1,2, Huaxiang Gu1,2, Yu Zhang1,2, Xun Li1,2, and Fei Wang1,2* 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 Revised: March 5, 2016 Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing Accepted: March 9, 2016 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 171 254 271 First published online activity by 79.5%. Three highly conserved residues (Asp , His , and His ) in TtGlyDH were March 14, 2016 associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA *Corresponding author reduction, TtGlyDH showed maximum activity towards glycerol at 60°C and pH 8.0 and Phone: +86-25-85427649; towards DHA at 60°C and pH 6.0. DHA reduction was the dominant reaction, with a lower Fax: +86-25-85427649; K of 1.08 ± 0.13 mM and V of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, E-mail: [email protected] m(DHA) max 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. pISSN 1017-7825, eISSN 1738-8872 Keywords: Dinucleotide binding, glycerol dehydrogenase, molecular modeling, site-directed Copyright© 2016 by The Korean Society for Microbiology mutagenesis, Thermoanaerobacterium thermosaccharolyticum and Biotechnology Introduction closely related to Fe-ADHs. However, most reported GlyDHs are NAD+-linked and strictly zinc-dependent metalloenzymes. In vivo glycerol metabolism involves many different Moreover, most studies on GlyDH have been conducted in kinds of proteins, among which glycerol dehydrogenase the thermophilic species B. stearothermophilus, due to the (GlyDH, E.C.: 1.1.1.6) primarily catalyzes the conversion of availability of its crystal structure [5, 30]. However, glycerol to 1,3-dihydroxypropranone (DHA) coupled with biochemical assays in this species are conducted at 30°C, the reduction of nicotinamide adenine dinucleotide (NAD+). lower than its optimal growth temperature of 55°C [21, 26]. GlyDHs effectively regulate physiological processes related To date, biochemical characterization of GlyDHs in to energy production, exchange, and consumption and have thermophiles has not been conducted at high temperatures. been isolated from a variety of prokaryotic and eukaryotic The Rossmann fold in dehydrogenases is one of the most cells, including Escherichia coli [27], Bacillus megaterium [22], common structural features of supersecondary structures Bacillus stearothermophilus [26], Clostridium butyricum [18], in many oxidoreductases that bind NAD+, nicotinamide and Schizosaccharomyces pombe [13]. Based on its PROSITE adenine dinucleotide phosphate (NADP+), and related description, there are three known types of alcohol cofactors. Since Rossmann first described the dinucleotide dehydrogenases (ADHs): zinc-containing “long-chain” alcohol binding fold in 1974 based on the structural alignment dehydrogenases (Zn-ADHs), insect-type or “short-chain” of four dehydrogenases (lactate, malate, alcohol, and alcohol dehydrogenases, and iron-containing alcohol glyceraldehyde-3-phosphate dehydrogenases) [20], a great dehydrogenases (Fe-ADHs). Of these, bacterial GlyDHs are deal of structural data on classical dinucleotide binding June 2016 ⎪ Vol. 26⎪ No. 6 1078 Wang et al. proteins have suggested that the initial βαβ fold is the most residue are critical for dinucleotide binding in TtGlyDH, conserved unit in the Rossmann fold [8] and typically based on rational design and site-directed mutagenesis. contains a phosphate binding motif (GXGXXG, where X is any amino acid) [3]. However, these traditional structural Materials and Methods features provide insufficient information to interpret the interactions between the Rossmann fold and nicotinamide Chemicals, Bacterial Strains, and Culture Conditions dinucleotides for all GlyDHs. All chemicals were purchased from Sangon Biotech (Shanghai, The lack of structural information on GlyDHs in China), unless otherwise stated. All DNA restriction enzymes, T4 thermophiles is the main bottleneck to determining the key polynucleotide kinase, and ligase were purchased from TaKaRa residues within its active and dinucleotide binding sites. To (Dalian, China). Phusion High-Fidelity DNA Polymerase was purchased from New England Biolabs (Ipswich, MA, USA). The date, only six delicate crystal structures of GlyDHs have BIOMEGA PCR Purification Kit and Mini Plasmid Extraction Kit been resolved and deposited in the Brookhaven Protein (Shanghai, China) were purchased for DNA purification and Data Bank (PDB), including three GlyDH structures from plasmid isolation. The genomic DNA from T. thermosaccharolyticum B. stearothermophilus (PDB entry: 1JQ5, 1JPU, and 1JQA) [5, DSM 571 was purchased from the German Collection of 21, 30] and one structure each from Thermotoga maritima Microorganisms and Cell Cultures (DSMZ; http://www.dsmz.de/). (PDB entry: 1KQ3) [11], Clostridium acetobutylicum (PDB E. coli TOP10 was used for plasmid propagation. E. coli BL21 entry: 3CE9), and Serratia plymuthica (PDB entry: 4MCA) (DE3) was used as the host for heterologous expression. The E. coli [14]. Moreover, the detailed mechanism of GlyDHs has strains were cultured at 37°C in LB medium supplemented with not been elucidated owing to the limitation of structural ampicillin (100 µg/ml) when required for plasmid maintenance. information and biochemical data. However, combining protein engineering techniques and protein structure Construction of Plasmids and Strains prediction could provide an alternative approach to DNA manipulations were performed by following standard procedures. A pair of specific oligonucleotide primers (see below) studying GlyDH function and mechanism. Rational design for amplifying gldA was designed based on its reference DNA has been performed with varying degrees of success to sequence (Gene ID: 9707383, Tthe_1821). The gldA coding sequence identify potential active residues and improve the given was amplified using genomic DNA from T. thermosaccharolyticum properties of enzymes based on in silico prediction and DSM 571 as a template: modeling. For example, redesigning the coenzyme specificity 5’-GGGAATTCCATATGACAAAAGCTATAATAGGCCCTTCG-3’ of a dehydrogenase using protein engineering was conducted (forward) in 1990 [24], and the coenzyme specificities of an ADH 5’-CCGCTCGAGTCTAGATCTCTTATTTTTGTACATTTTTCC-3’ from Rana perezi [19] and a xylitol dehydrogenase from (reverse) Pichia stipitis [29] were reversed completely by substituting The underlined letters represent the NdeI and XhoI restriction the key residues necessary for coenzyme binding, using the sites, respectively. The integrity and yield of the PCR products assistance of structural simulation. were assessed using agarose gel electrophoresis. The resulting Compared with mesophiles, thermophile-derived enzymes PCR fragments were digested with NdeI and XhoI and ligated into the commercial vector pET-20b in frame with the His tag. are relatively thermostable and tolerant to industrial 6 The ligation mixtures were transformed into E. coli TOP10 by heat production conditions such as high salt or solvent shocking the chemically competent cells for 90 sec at 42°C without concentrations and high operation temperatures [4, 25, 28], shaking. The transformants were screened on LB plates containing and considerable research efforts in recent years have 100 µg/ml ampicillin. The transformed gldA was confirmed by focused on thermostable enzymes. Thermoanaerobacterium DNA sequencing. thermosaccharolyticum is a thermophilic obligate anaerobic bacterium, and the genomic data from T. thermosaccharolyticum Expression and Purification of Recombinant TtGlyDH DSM 571 are accessible from the GenBank database [6, 16]. Expression strain BL21 (DE3) cells harboring pET-20b-gldA However, there have been few reports on the biochemical were grown in 50 ml of LB (100 µg/ml ampicillin) at 37°C and or structural characterization of T. thermosaccharolyticum 180 rpm. IPTG was added to a final concentration of 0.5 mM to GlyDH. induce expression until the culture reached the stationary phase (OD = 0.6–0.8) and was incubated for an additional 3 h at 25°C In this study, we examined a putative GlyDH from 600 at 120 rpm. The cultured cells were harvested and centrifuged at T. thermosaccharolyticum DSM 571 (TtGlyDH) and described 4°C and 10,000 ×g for 5 min. The cell pellets were washed twice its cloning, expression, and biochemical characterization. with ice-cold water to remove residual medium and suspended in We found that the GGG motif and a basic amino acid 5 ml of binding buffer (5 mM imidazole,