Correspondence: Jaeho Cha, Department of Microbiology, Pusan National University, Busandaehak-ro 63beon-gil 2, Geumjeong-gu, Busan 609-735, Korea. Tel.: +82 51 510 2196; fax: +82 51 514 1778; e-mail: email@example.com
4-α-Glucanotransferase, an enzyme encoded by malQ, transfers 1,4-α-glucan to an acceptor carbohydrate to produce long linear maltodextrins of varying lengths. To investigate the biochemical characteristics of the malQ gene (Sde0986) from Saccharophagus degradans 2-40 and to understand its physiological role in vivo, the malQ gene was cloned and expressed in Escherichia coli. The amino acid sequence of MalQ was found to be 36–47% identical to that of amylomaltases from gammaproteobacteria. MalQ is a monomeric enzyme that belongs to a family of 77 glycoside hydrolases, with a molecular mass of 104 kDa. The optimal pH and temperature for MalQ toward maltotriose were determined to be 8.5 and 35 °C, respectively. Furthermore, the enzyme displayed glycosyl transfer activity on maltodextrins of various sizes to yield glucose and long linear maltodextrins. MalQ, however, could be distinguished from other bacterial and archaeal amylomaltases in that it did not produce maltose and cyclic glucan. Reverse transcription PCR results showed that malQ was not induced by maltose and was highly expressed in the stationary phase. These data suggest that the main physiological role of malQ in S. degradans is in the degradation of glycogen, although the gene is commonly known to be involved in maltose metabolism in E. coli.
The enzyme 4-α-glucanotransferase (MalQ, EC 22.214.171.124) releases the reducing end glucosyl units from α-1,4-glucan and transfers the nonreducing dextrinyl moiety onto glucose or another α-1,4-glucan. It was first discovered in bacteria and designated as amylomaltase (Monod & Torriani, 1948). Similar enzyme activity was also reported in plants by an enzyme called disproportionating enzyme (D-enzyme) (Jones & Whelan, 1969). However, in eukaryotic cells, glycogen-debranching enzyme (GDE or AGL) performs a dual role by functioning as a glucosyltransferase and a glucosidase (Nakayama et al., 2001). In general, MalQ can produce either linear α-1,4-glucans, consisting of 40 glucose units with maltooligosaccharides, or cycloamyloses that consist of amylose with a degree of polymerization (DP) ranging from 17 to > 100 with amylose (Takaha et al., 1996).
It has been reported that MalQ plays an important role in maltose/maltodextrin metabolism (Boos & Shuman, 1998; Dippel & Boos, 2005). MalQ releases glucose and maltodextrin, and then, the former enters the glycolysis pathway, whereas the latter is degraded by MalP (maltodextrin phosphorylase) and MalZ (maltodextrin glucosidase) to produce energy. Thus, in this process, MalQ and MalZ use maltotriose as the smallest substrate, whereas MalP does not. The smallest substrate for MalP is maltotetraose. The growth restriction of a malQ deletion strain in maltose medium demonstrates the importance of MalQ in maltose metabolism (Schwartz, 1967; Park et al., 2011).
Recently, it has been reported that MalQ is involved in glycogen formation (Park et al., 2011). MalQ acts on short maltodextrins and generates high-DP maltodextrins, which serve as substrates for GlgB (branching enzyme). Moreover, MalQ can carry out the resynthesis of glycogen. Maltotriose and maltotetraose are endogenous inducers produced by the sequential catalytic activity of GlgP (glycogen phosphorylase) and GlgX (debranching enzyme) on glycogen, followed by disproportionation of glycogen by MalQ to form a precursor for glycogen.
Saccharophagus degradans 2-40, an aerobic, gram-negative, marine gammaproteobacterium, has been studied to elucidate its mechanism of degradation and production of energy sources from complex polysaccharides, including agar and cellulose (Taylor et al., 2006; Kim et al., 2010; Shin et al., 2010). However, the enzymes related to the energy storage mechanism in vivo remain to be identified. In this study, the biochemical properties of MalQ in S. degradans were characterized, and its potential role in glycogen degradation was assessed by reverse transcription PCR analysis.
Materials and methods
Strains and growth conditions
Escherichia coli BL21(DE3) cells were grown in LB broth (1% tryptone, 0.5% yeast extract, and 1% NaCl) at 37 °C on a shaker (250 rpm). The growth medium was supplemented with kanamycin (50 μg mL−1) as needed. S. degradans 2-40 was grown in 2.3% Instant Ocean Sea Salt (Aquarium Systems, Mentor, OH), 0.1% yeast extract, 0.05% ammonium chloride, 50 mM Tris–HCl (pH 7.4), and 0.2% or 0.5% of glucose, maltose, and xylose at 30 °C on a shaker (250 rpm).
Cloning of malQ from S. degradans 2-40
The malQ gene was amplified from the chromosomal DNA of S. degradans using Sde4GT-for and Sde4GT-rev primers (Table S1). The 2,175-bp PCR product was purified and digested with restriction enzymes (NdeI and XhoI). The purified DNA fragment was inserted into a protein expression vector, pET29b+, to create a C-terminal 6× histidine fusion for purification. All cloning kits and enzymes were used according to the manufacturer's instructions (Takara Bio Inc., Otsu, Japan).
Purification of MalQ
The E. coli BL21(DE3) strain harboring pET29b-Sde4GT was incubated at 37 °C on a shaker (250 rpm). For induction, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added when the optical density (600 nm) reached 0.6–0.8. After additional incubation at 16 °C for 18 h on a shaker (250 rpm), cells were harvested and washed twice with ice-cold Buffer A (20 mM sodium phosphate [pH 7.4], 0.5 M NaCl) and disrupted by a French pressure cell press (Thermo Electron Co., Waltham, MA). Cell debris was removed by centrifugation (13 000× g, 30 min, 4 °C), and the supernatant containing MalQ was purified by Ni-nitrilotriacetic acid affinity chromatography. To achieve better expression, low-temperature induction (16 °C) was performed because considerable amounts of inclusion bodies were formed (data not shown). All materials and buffers were kept at 4 °C for purification of MalQ using Ni-NTA chromatography because MalQ was extremely sensitive to warm temperatures. Subsequently, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out to visualize the purified protein.
Biochemical properties of MalQ
One microgram of enzyme was mixed with 50 μL of 0.2% (w/v) maltotriose dissolved in 25 mM Tris-HCl (pH 8.5) and incubated at 35 °C for 2 min. The reaction mixture was boiled for 10 min to inactivate the enzyme. After centrifugation (13 000× g, 5 min at 4 °C), 50 μL of the reaction mixture was added to 100 μL of the glucose oxidase/peroxidase reagent (purchased from Sigma-Aldrich, St. Louis, MO) and incubated at 37 °C for 30 min. Subsequently, 100 μL of 12 N H2SO4 was added to the sample, and the absorbance was measured at 540 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of glucose per minute.
The effect of temperature on enzyme activity was determined by heating the samples at 10–65 °C. The optimal pH for the enzyme was determined to be 35 °C in a pH range of 4.0–12.0 using the following buffer systems: 25 mM sodium acetate (pH 4.0–6.0), sodium phosphate (pH 6.0–7.0), Tris–HCl (pH 7.0–9.0), and glycine–NaOH (pH 9.0–12.0). All the mixtures (0.05 mL) containing 1.1 μg of enzyme and 0.2% (w/v) maltotriose were incubated for 2 min. The absorbance of released glucose was measured at 540 nm, and the relative activity was expressed as a percentage of the maximum enzyme activity. All experiments were carried out in triplicate.
To identify the reaction products by MalQ, the reaction mixture (0.05 mL) containing 0.2% (w/v) maltotriose in 25 mM Tris–HCl (pH 8.5) and 1 μg enzyme was incubated at 35 °C for 4 h and then treated with glucoamylase (16 U) at 50 °C for 8 h. The reaction products were examined by thin layer chromatography.
Thin layer chromatography
The reaction products of polysaccharides with 4-α-glucanotransferase were spotted on a silica gel plate (Whatman, Dassel, Germany). The plate was placed in a chamber filled with a solvent mixture of 1-butanol/ethanol/distilled water (5 : 3 : 2 [v/v/v]) and was developed by dipping it into a solution containing 0.3% (w/v) naphthol and 5% (v/v) H2SO4, followed by baking at 120 °C for 10 min.
Isolation and quantification of glycogen
Saccharophagus degradans was grown at 30 °C on a shaker (250 rpm), and 200 mL was removed at various time points (9, 10, and 16 h after inoculation) during the exponential phase with 0.5% (w/v) glucose, maltose, and xylose as the carbon sources. Samples were also collected at various time points (29, 22, and 32 h after inoculation) during the stationary phase. The culture was then harvested and washed twice with Buffer A. Pellets were resuspended in 0.05 volume of 200 mM sodium acetate (pH 4.5) and sonicated. Cell debris was removed by centrifugation (13 000× g, 30 min, 24 °C), and the supernatant was divided into two aliquots for the analyses of protein and glycogen. The glycogen assay was performed according to a previously described method (Dauvillee et al., 2005). Sixteen units of amyloglucosidase (purchased from Sigma-Aldrich) were added to 100 μL of the crude extract and incubated at 50 °C for 30 min. Subsequently, the reaction mixture was incubated at 95 °C for 10 min to quench the enzyme activity. As previously described, the released glucose from glycogen was measured. Varying glycogen concentrations (0–2.5 μg) were used as standards to generate the standard curve.
RNA preparation and quantitative RT-PCR
Saccharophagus degradans was harvested after growth in a medium containing 0.5% glucose, maltose, and xylose to isolate total RNA at the exponential and stationary phases. RNA was purified using the Total RNA extraction kit (Intron Co., Daejeon, Korea) according to the manufacturer's instructions. cDNA was prepared from 150 ng of total RNA with a random hexamer using a cDNA Synthesis kit (Fermentas, Ontario, Canada). cDNA was then used as a template for RT-PCR with the primer pairs listed in Table S1. Guanylate kinase (spoR) was used as a reference gene (Zhang & Hutcheson, 2011).
One-way analysis of variance (anova) test was applied to compare the groups using Prism 5 (Graphpad Software, Inc., San Diego, CA). P values < 0.05 were considered to be significant.
Results and discussion
Genetic location and sequence comparison of MalQ with 4-α-glucanotransferases from various organisms
When the amino acid sequence was analyzed by a blast search, Sde0986, annotated as a 4-α-glucanotransferase, was selected as the candidate for encoding MalQ (Weiner et al., 2008). The putative malQ gene of S. degradans had relatively low amino acid sequence identity with its homologous genes in other gammaproteobacteria such as Teredinibacter turnerae (47%) and E. coli (36%).
Most MalQs were classified as part of the glycoside hydrolase (GH) family 77 because of their structure: the core structure consisted of a (β/α)8 barrel based on the amino acid sequence (Coutinho & Henrissat, 1999). GH77 belongs to the α-amylase superfamily (Clan H) along with GH13 and GH70. Similarly, GH13 has 4 conserved domains that contribute to its catalytic activity, whereas the structural specificity in the conserved residues has been identified in GH77 enzymes (Przylas et al., 2000). Three important acidic residues could be found in MalQ of S. degradans at Asp480, Glu528, and Asp580 (Fig. 1a), similar to the residues in Taka-amylase A of Aspergillus sp., Asp206, Glu230, and Asp297 that serve as a nucleophile, a proton donor, and a stabilizer in the transition state, respectively (MacGregor et al., 2001).
Unlike the malPQ operon in E. coli, which is located far from the glycogen operon, the malQ of S. degradans was adjacent to a putative glycogen operon (Fig. 1b). A homolog of the expected MalP was absent. Furthermore, the full genome sequence of S. degradans showed no genes homologous to malEFG of E. coli (Weiner et al., 2008). The same gene arrangement with a putative glycogen operon of S. degradans was found in Alteromonadales. These observations indicate that S. degradans malQ is not involved in maltose catabolism, but it is involved in glycogen metabolism instead.
Expression and purification of MalQ
Approximately 8.5 mg of pure recombinant MalQ was obtained from 1 L of cell culture. The size of MalQ estimated by computation of the theoretical calculation (http://web.expasy.org/compute_pi/) and SDS-PAGE analysis was approximately 82 kDa (Fig. S1a). The molecular mass of MalQ, as determined by gel filtration chromatography using a Superdex 200 column (GE Healthcare), was approximately 104 kDa (Fig. S1b), suggesting that MalQ is a monomeric enzyme.
Biochemical properties and mechanism of action of MalQ
Temperature and pH activity profiles of MalQ were analyzed by detecting the amount of released glucose. At 35 °C, MalQ activity was determined at pH 4.0–12.0, and relatively, high activity (> 60%) was noted at pH 7.0–9.0; the maximum activity was detected at pH 8.5 (Fig. 2a). At the optimal pH, the enzyme activity was examined at various temperatures, ranging from 10 to 65 °C, and the highest enzyme activity was detected at 35 °C (Fig. 2b). These are typical features of enzymes found in a mesophilic organism (Kitahata et al., 1989). Purified MalQ was tested for its activity on various types of sugars to identify its substrate specificity. MalQ activity was detected on various glucose polysaccharides from maltotriose to maltoheptaose. However, no glycosyltransferase activity was detected on glucose and maltose (Fig. 3a). The 4-α-glucanotransferase derived from plants such as Solanum tuberosum (potato) produces glucose, maltotriose, and maltodextrins (≥G4), but not maltose (Takaha & Smith, 1999). The activity of the MalQ from S. degradans was similar to that of D-enzyme from potato in that there was no maltose production during the enzyme reaction on any substrate (Fig. 3). In addition, it has been reported that the 4-α-glucanotransferase from S. tuberosum, E. coli, Bacillus spp., Thermus aquaticus, and Thermococcus litoralis could produce cycloamyloses with amylose (Takaha & Smith, 1999). We examined whether S. degradans MalQ could produce cyclic glucans. The reaction products by MalQ were not hydrolyzed by glucoamylase treatment, indicating that the products were linear (Fig. S2).
4-α-Glucanotransferases require an acceptor to break down complex polysaccharides (Boos & Shuman, 1998). To confirm this, S. degradans MalQ was incubated with amylose (α-1,4-glucan), starch, pullulan, and glycogen (α-1,4- and α-1,6-glucan) in the presence of glucose or maltose as an acceptor. As shown in Fig. 3b, glycosyltransferase activity was detected in the amylose or starch containing glucose and maltose. Generally, both glucose and maltose are acceptors against MalQ, but glucose is a more efficient acceptor than maltose (Jung et al., 2010). The final products were glucose, maltotriose, and maltodextrins (≥G4). Pullulan and glycogen were not hydrolyzed despite the presence of glucose. Based on the biochemical characteristics described above, we conclude that S. degradans MalQ is a type II 4-α-glucanotransferase that uses a maltotriose as the smallest donor molecule, glucose as the smallest acceptor, and maltose as the smallest transferred unit.
Long linear maltodextrin production by MalQ is involved in glycogen degradation
The relationship between glycogen and MalQ levels was evaluated. S. degradans was grown in the medium containing 0.5% glucose, maltose, or xylose to measure the glycogen content. The glycogen content during the exponential phase was 0.31, 0.38, and 0.14 mg mg−1 protein with glucose, maltose, and xylose used as the carbon source, respectively. In contrast, greater amounts of glycogen (from 3.07 to 0.75 and 0.98 mg mg−1 protein) were measured during the stationary phase. Notably, glycogen in S. degradans accumulated in the stationary phase such that the amounts were threefold greater in the glucose medium than those in the maltose or xylose medium (Fig. 4a).
To examine the relationship between the glycogen content and malQ expression, RT-PCR was employed. During the exponential phase, the expression level of glgA, which encodes a glycogen synthase, was not significantly different under the tested conditions, whereas the putative amylase gene (amy, sde0563) was highly induced by maltose (Fig. 4b). The expression of malQ was not induced by any type of sugar assessed in this study and was maintained at low levels. At the stationary phase, malQ expression was highly increased in the glucose medium, whereas glgA and amy expression levels were not significantly different in three carbon sources (Fig. 4c). The role of MalQ in E. coli has been extensively studied (Schwartz, 1987; Decker et al., 1993; Boos & Shuman, 1998; Dippel & Boos, 2005). The genes involved in maltose utilization, including malQ, are induced by all maltodextrins in the medium serving as a carbon source in vivo. For maltodextrin metabolism, MalQ and MalP, via maltodextrin intermediates, yields glucose and glucose-1-P that enter glycolysis. MalQ is also involved in glycogen metabolism. GlgP and GlgX lead to the formation of maltodextrins from glycogen, and the internal maltodextrins are further degraded by MalP and MalZ with the help of MalQ (Dippel et al., 2005). Based on the RT-PCR results, we suggest that MalQ does not play a role in maltose utilization, rather it may act to metabolize the internal maltodextrins formed during glycogen degradation in S. degradans.
We thank Prof. Kyoung Heon Kim (Korea University, Republic of Korea) for providing the Saccharophagus degradans 2-40 strain. This work was supported by the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation, and Maritime Affairs.