Hyunju Cha

Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance

ABSTRACT The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.

Structural Insight Into the Bifunctional Mechanism of the Glycogen-Debranching Enzyme Trex from the Archaeon Sulfolobus Solfataricus.

TreX is an archaeal glycogen-debranching enzyme that exists in two oligomeric states in solution, as a dimer and tetramer. Unlike its homologs, TreX from Sulfolobus solfataricus shows dual activities for α-1,4-transferase and α-1,6-glucosidase. To understand this bifunctional mechanism, we determined the crystal structure of TreX in complex with an acarbose ligand. The acarbose intermediate was covalently bound to Asp363, occupying subsites -1 to -3. Although generally similar to the monomeric structure of isoamylase, TreX exhibits two different active-site configurations depending on its oligomeric state. The N terminus of one subunit is located at the active site of the other molecule, resulting in a reshaping of the active site in the tetramer. This is accompanied by a large shift in the “flexible loop” (amino acids 399-416), creating connected holes inside the tetramer. Mutations in the N-terminal region result in a sharp increase in α-1,4-transferase activity and a reduced level of α-1,6-glucosidase activity. On the basis of geometrical analysis of the active site and mutational study, we suggest that the structural lid (acids 99-97) at the active site generated by the tetramerization is closely associated with the bifunctionality and in particular with the α-1,4-transferase activity. These results provide a structural basis for the modulation of activities upon TreX oligomerization that may represent a common mode of action for other glycogen-debranching enzymes in higher organisms.

TreX from Sulfolobus solfataricus ATCC 35092 Displays Isoamylase and 4-α-Glucanotransferase Activities

A treX in the trehalose biosynthesis gene cluster of Sulfolobus solfataricus ATCC 35092 has been reported to produce TreX, which hydrolyzes the α-1,6-branch portion of amylopectin and glycogen. TreX exhibited 4-α-D-glucan transferase activity, catalyzing the transfer of α-1,4-glucan oligosaccharides from one molecule to another in the case of linear maltooligosaccharides (G3–G7), and it produced cyclic glucans from amylopectin and amylose like 4-α-glucanotransferase. These results suggest that TreX is a novel isoamylase possessing the properties of 4-α-glucanotransferase.

Oligomeric and functional properties of a debranching enzyme (TreX) from the archaeon Sulfolobus solfataricus P2

A gene, treX, encoding a debranching enzyme previously cloned from the trehalose biosynthesis gene cluster of Sulfolobus solfataricus P2 was expressed in Escherichia coli as a His-tagged protein and the biochemical properties were studied. The specific activity of the S. solfataricus debranching enzyme (TreX) was highest at 75°C and pH 5.5. The enzyme exhibited hydrolysing activity toward α-1,6-glycosidic linkages of amylopectin, glycogen, pullulan, and other branched substrates, and glycogen was the preferred substrate. TreX has a high specificity for hydrolysis of maltohexaosyl α-1,6-β-cyclodextrin, indicating the high preference for side chains consisting of 6 glucose residues or more. The enzyme also exhibited 4-α-sulfoxide-glucan transferase activity, catalysing transfer of α-1,4-glucan oligosaccharides from one chain to another. Dimethyl sulfoxide (10%, v/v) increased the hydrolytic activity of TreX. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation revealed that the enzyme exists mostly as a dimer at pH 7.0, and as a mixture of dimers and tetramers at pH 5.5. Interestingly, TreX existed as a tetramer in the presence of DMSO at pH 5.5–6.5. The tetramer showed a 4-fold higher catalytic efficiency than the dimer. The enzyme catalysed not only intermolecular trans-glycosylation of malto-oligosaccharides (disproportionation) to produce linear α-1,4-glucans, but also intramolecular trans-glycosylation of glycogen. The results presented in this study indicated that TreX may be associated with glycogen metabolism by selective cleavage of the outer side chain.

Modulation of Substrate Preference of Thermus Maltogenic Amylase by Mutation of the Residues at the Interface of a Dimer

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k cat/K m value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.

Dissociation/association properties of a dodecameric cyclomaltodextrinase : Effects of pH and salt concentration on the oligomeric state

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I‐5 (CDase I‐5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I‐5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain‐swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I‐5 was present as a dimer in the pH range of 5.0–6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I‐5 dissociated into dimers at a rate of 8.58 × 10−2 h−1 at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild‐type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH‐dependent process, the dissociation of wild‐type CDase I‐5 proceeded very fast at pH 7.0 in the presence of 0.2–1.0 m of KCl. Stopped‐flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s−1 and 7.99 s−1 in the presence of 0.2 m and 1.0 m of KCl, respectively.

Thermostable and alkalophilic maltogenic amylase of Bacillus thermoalkalophilus ET2 in monomer-dimer equilibrium

A gene encoding a thermostable and alkalophilic maltogenic amylase (BTMA) was cloned from the thermophilic bacterium Bacillus thermoalkalophilus ET2. BTMA was composed of 588 amino acids with a predicted molecular mass of 68.8 kDa. The enzyme had an optimal temperature and pH of 70°C and 8, respectively, the highest among maltogenic amylases reported so far. The Tm of BTMA at pH 8 was 76.7°C with an enthalpy of 113.6 kJ mol−1. Both hydrolysis and transglycosylation activities for various carbohydrates were evident. β-Cyclodextrin (β-CD) and soluble starch were hydrolyzed mainly to maltose, and pullulan to panose. Acarbose, a strong amylase inhibitor, was hydrolyzed by BTMA to glucose and acarviosine-glucose. The Km and kcat values of BTMA for β-CD hydrolysis were 0.128 mM and 165.8 s−1 mM, respectively. The overall catalytic efficiency (kcat/Km) of the enzyme was highest toward β-CD. BTMA was present in a monomer-dimer equilibrium with a molar ratio of 54:46 in 50 mM glycine-NaOH buffer (pH 8.0). This equilibrium could be affected by KCl and enzyme concentrations. The multi-substrate specificity of the enzyme was modulated by the structural differences between monomeric and dimeric forms. Starch was hydrolyzed more readily when monomeric BTMA was prevalent, while the opposite was observed for β-CD.

Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu.

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with Km=0.38mM and kcat/Km=20.58mM(-1)s(-1) for hydrolysis, and Km2=18.38mM and kcat2/Km2=2.57mM(-1)s(-1) for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235-located on a wide open groove near subsite +1-is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.

Preliminary X-ray Crystallographic Analysis of a Novel Maltogenic Amylase From Bacillus Stearothermophilus ET1

A novel maltogenic amylase from Bacillus stearothermophilus ET1, which has a dual activity of alpha-1,4- and alpha-1,6-glycosidic bond cleavages and alpha-1,6-glycosidic bond formation, was crystallized by using the hanging-drop vapor-diffusion method. The best crystals were obtained by employing a high concentration of protein (56 mg ml-1) and a precipitant containing 22% glycerol, 1.6 M ammonium sulfate in 0.1 M Tris-HCl (pH 8.5). Native diffraction data to 2.66 A resolution have been obtained from crystals flash-frozen at 110 K. The crystals belong to the space group P212121 with unit-cell dimensions of a = 77.62, b = 121.23, c = 244. 29 A, and contain three or four protomers per asymmetric unit. Structure determination by multiple isomorphous replacement is in progress.

Structural Feature of the Archeal Glycogen Debranching Enzyme from Sulfolobus Solfataricus

TreX is an archaeal glycogen debranching enzyme which catalyses both the α-1, 4- transferase and α-1, 6-glucosidase activity, similar to GDEs in mammals and yeast. It exists in two oligomeric states in a solution, as a dimer and a tetramer, with its tetramer showing a four-fold higher catalytic efficiency compared to that of the dimer. TreX has a high specificity for hydrolysis of maltohexaosyl α-1, 6-β-cyclodextrin, showing the high preference for side chains consisting of 6 glucose residues or more. The structure of TreX reveals the unique arrangement of the subunits with the substrate binding grooves connected each other in tetrameric form, adopting a suitable architecture for binding to the branched glycogen. The analysis of the active cleft shows that the helix at the substrate binding groove provides a platform for the stable binding to the longer substrate, explaining the substrate specificity of TreX. Based on the structural analysis and biochemical study, we suggest that the unique dual catalytic property of the archaeal debranching enzyme may be associated to the tetramer, giving rise to the modulation of the activities of TreX upon oligomerization of its subunits.