Structural Insight into the Bifunctional Mechanism of the Glycogen-debranching Enzyme TreX from the Archaeon Sulfolobus solfataricus

Abstract

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 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. Debranching enzymes are divided into two groups. One includes isoamylase and pullulanase found in bacteria and plants, and the other includes glycogen-debranching enzymes (GDEs) 4The abbreviations used are: GDE, glycogen-debranching enzyme; aa, amino acids; CHES, 2-(cyclohexylamino)ethanesulfonic acid. 4The abbreviations used are: GDE, glycogen-debranching enzyme; aa, amino acids; CHES, 2-(cyclohexylamino)ethanesulfonic acid. found in mammals and yeast. Whereas the former has only a single α-1,6-glycosidic bond hydrolyzing activity for glycogen and amylopectin, the GDEs in eukaryotes are bifunctional, possessing both α-1,4-transferase (EC 2.4.1.25) and α-1,6-glucosidase (EC 3.2.1.33) activities within a single polypeptide chain (1Bates E.J. Heaton G.M. Taylor C. Kernohan J.C. Cohen P. FEBS Lett. 1975; 58: 181-185Crossref PubMed Scopus (58) Google Scholar, 2Nakayama A. Yamamoto K. Tabata S. J. Biol. Chem. 2001; 276: 28824-28828Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The structure of GDE in eukaryotes has yet to be determined. TreX is an archaeal enzyme in Sulfolobus that debranches the side chain of glycogen into maltodextrin, which is further converted to trehalose (3Maruta K. Hattori K. Nakada T. Kubota M. Sugimoto T. Kurimoto M. Biochim. Biophys. Acta. 1996; 1289: 10-13Crossref PubMed Scopus (60) Google Scholar). It belongs to the GH13 (glycoside hydrolase 13) family and shows high sequence similarity to isoamylase and pullulanase. The amino acid sequence of TreX shows 74% homology to isoamylase from Sulfolobus acidocaldarium, 42% homology to the glycogen operon protein GlgX from Escherichia coli, and 30% homology to isoamylase from Pseudomonas. However, despite its high sequence similarity to isoamylase, which has only a single α-1,6-glycosidic bond hydrolyzing activity, TreX exhibits α-1,4-transferase as well as α-1,6-glucosidase activity, similar to GDEs in mammals and yeast (4Park H.S. Park J.-T. Kang H.K. Cha H. Kim D.S. Kim J.W. Park K.-H. Biosci. Biotechnol. Biochem. 2007; 71: 1348-1352Crossref PubMed Scopus (31) Google Scholar, 5Park J.-T. Park H.S. Kang H.K. Hong J.S. Cha H. Woo E.-J. Kim J.W. Kim M.J. Boos W. Lee S. Park K.-H. Biocatal. Biotransform. 2008; 26: 76-85Crossref Scopus (17) Google Scholar). To date, it is the only GDE found in bacteria/archaea with α-1,4-transferase activity, transferring the α-1,4-glucan oligosaccharides from one molecule to another using various substrates, including glycogen and amylopectin (4Park H.S. Park J.-T. Kang H.K. Cha H. Kim D.S. Kim J.W. Park K.-H. Biosci. Biotechnol. Biochem. 2007; 71: 1348-1352Crossref PubMed Scopus (31) Google Scholar). In a previous study, we cloned the gene treX from the trehalose biosynthesis gene cluster of Sulfolobus solfataricus P2, a hyperthermophilic archaeon, and expressed the protein in E. coli (4Park H.S. Park J.-T. Kang H.K. Cha H. Kim D.S. Kim J.W. Park K.-H. Biosci. Biotechnol. Biochem. 2007; 71: 1348-1352Crossref PubMed Scopus (31) Google Scholar). The treX gene is one of three trehalose operon genes, including treX, treY (maltooligosyltrehalose synthase), and treZ (maltooligosyltrehalose trehalohydrolase) (3Maruta K. Hattori K. Nakada T. Kubota M. Sugimoto T. Kurimoto M. Biochim. Biophys. Acta. 1996; 1289: 10-13Crossref PubMed Scopus (60) Google Scholar, 6Maruta K. Kubota M. Fukuda S. Kurimoto M. Biochim. Biophys. Acta. 2000; 1476: 377-381Crossref PubMed Scopus (19) Google Scholar, 7Maruta K. Mitsuzumi H. Nakada T. Kubota M. Chaen H. Fukuda S. Sugimoto T. Kurimoto M. Biochim. Biophys. Acta. 1996; 1291: 177-181Crossref PubMed Scopus (102) Google Scholar). TreX exists in two oligomeric states in solution, as a dimer and tetramer. The tetramer formed in the presence of dimethyl sulfoxide shows a 4-fold higher catalytic efficiency compared with the dimer (5Park J.-T. Park H.S. Kang H.K. Hong J.S. Cha H. Woo E.-J. Kim J.W. Kim M.J. Boos W. Lee S. Park K.-H. Biocatal. Biotransform. 2008; 26: 76-85Crossref Scopus (17) Google Scholar). However, the mechanism underlying the increase in activity upon tetrameric rearrangement is not clearly understood. To elucidate the modulation of activity upon oligomerization and the mechanism of the dual functions of the enzyme, we carried out a structural study on the GDE TreX. In this study, we describe the crystal structure of the archaeal GDE TreX from S. solfataricus at a resolution of 2.8 Å in complex with an acarbose ligand. Analysis of the structure revealed the unique tetrameric configuration of TreX reshaping the active site with an additional region from the N-terminal domain. This result provides a structural basis for the modulation of activity upon oligomerization and for the bifunctional mechanism of the archaeal GDE TreX. Protein Purification—E. coli cells carrying pSGDE were cultured in LB medium plus ampicillin and harvested by centrifugation. The cells were resuspended in lysis buffer (50 mm Tris-HCl (pH 7.5), 300 mm NaCl, and 10 mm imidazole) and sonicated using a VC-600 sonicator (Sonics & Materials, Inc., Newtown, CT). The crude cell extract was centrifuged at 10,000 × g for 15 min at 4 °C. After sonication, the cell lysates were subjected to nickel-nitrilotriacetic acid affinity chromatography (Qiagen) as described (8Kim T.J. Kim M.J. Kim B.C. Kim J.C. Cheong T.K. Kim J.W. Park K.-H. Appl. Environ Microbiol. 1999; 65: 1644-1651Crossref PubMed Google Scholar). α-1,4-Transferase Activity—The disproportionation activity of TreX and its mutants was determined by a glucose oxidase/peroxidase method (9Fox J.D. Robyt J.F. Anal. Biochem. 1991; 195: 93-96Crossref PubMed Scopus (361) Google Scholar) with slight modification. The reaction mixture contained 800 μl of glucose oxidase solution from a glucose kit (Youngdong Pharmaceutical Co., Seoul, Korea) and 1 μm enzyme in 50 mm sodium acetate buffer (pH 5.5). This reaction mixture was incubated at 40 °C for 10 min. The reaction was started by adding 40 μl of substrate (10 and 20 mm maltotriose). The absorbance of the reaction mixture was measured for 30 min at 500 nm. The equivalent of the hydrolyzed glycosidic bonds was based on glucose equivalents using a glucose equilibration curve. Enzyme activity was defined as the amount of glucose that 1 μm enzyme liberated per min. α-1,6-Glucosidase Activity—The α-1,6-hydrolysis activity of TreX and its mutants was assayed at 40 and 70 °C in 50 mm sodium acetate buffer (pH 5.5) plus glycogen by determining the reducing power of the hydrolysis products using the dinitrosalicylic acid method (10Miller G.L. Anal. Chem. 1959; 31: 426-428Crossref Scopus (22215) Google Scholar). One unit of enzyme was defined as the micromoles of maltose equivalents produced by 1 ml of enzyme/1 min. The specific activity (units/mg of enzyme) of each enzyme was calculated using the enzyme units. The enzyme reaction mixture (500 μl) was composed of 0.5% (w/v) glycogen (final concentration) as the substrate and 1 μm enzyme solution (final concentration) in 50 mm sodium acetate buffer (pH 5.5). The enzyme solution was added to the reaction and incubated for 10 min before the reaction was stopped by the addition of 500 μl of dinitrosalicylic acid solution. A reaction without addition of enzyme was used as a control. The reaction mixture was boiled for 5 min and cooled by placing the tube under running water. Absorbance was measured at 575 nm. The micromole equivalent of split bonds was based on the micromole of maltose equivalents using a maltose calibration curve. Crystallization and Data Collection—Crystallization trials of TreX were set up using the microbatch method under oil at 18 °C. The native protein was crystallized in the two different crystal forms, the dimeric and tetrameric forms, in space groups P3121 and P321, respectively. In both crystal forms, the asymmetric unit consisted of one dimer. The dimeric form of the TreX crystal was grown from 16% polyethylene glycol 8000, 0.2 m NaCl, and 0.1 m CHES buffer (pH 9.5). The tetrameric form of the crystal was grown from 2.2 m ammonium phosphate and 0.1 m Tris-HCl buffer (pH 8.5). For the acarbose intermediate complex crystal, 0.1% acarbose (kindly provided by Professor Seiya Chiba, Hokkaido University) was added to the protein, followed by incubation for 1 h prior to the setup of the crystal in 8% polyethylene glycol 3000, 0.2 m lithium sulfate, and 0.1 m imidazole buffer (pH 8.0). Before data collection, the crystals were cryocooled to 95 K using a cryoprotectant consisting of mother liquor supplemented with 20% glycerol. For the acarbose crystal, 0.1% acarbose was added to the cryoprotectant. The native and ligand-bound data sets were collected at the MX4II and MX6C beamlines of the Pohang Accelerator Laboratory, respectively. Structure Determination and Refinement—The diffraction data were processed and scaled with the program HKL2000 (11Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The native structure was determined by the molecular replacement method using the Phaser CCP4 suite (12Otwinowski Z. Minor W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) and the structure of Pseudomonas isoamylase (Protein Data Bank code 1BF2) as a search model. The resulting model was refined in conjunction with model rebuilding using the program CNS (13Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar). The structure of the complex was solved by direct refinement of the structure of native TreX against the diffraction data. The program Coot (14Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23224) Google Scholar) was used for manual model building, and the stereochemical quality of the model was monitored using the program PROCHECK (15Laskowski R.A. Moss D.S. Thornton J.M. J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1080) Google Scholar). The current atomic model of a native TreX dimer contains residues 9-718 with disordered N-terminal tails (residues 1-8) and has an R factor of 20.6% (Rfree = 23.1%). The data from the native crystal in the P321 space group were initially collected to 2.5-Å resolution. However, because of the presence of pseudo-merohedral twinning in the crystal, it was refined to only 3 Å, and the final R factors were relatively high in comparison with the resolution of the data. Molecular images were created using the program PyMOL (16DeLano W.L. The PyMOL Molecular Graphic System. DeLano Scientific, San Carlos, CA2002Google Scholar), and electron density maps were generated in Coot. The statistics for the diffraction data and the final refined structures are given in Table 1.TABLE 1Data and structure solution cell = = = = = = = = = in are statistics from the resolution in are statistics from the resolution in are statistics from the resolution = is the of and is the of refined factor = and are the and calculated structure factor respectively. was calculated using of the bond bond The in are statistics from the resolution = is the of and is the R factor = and are the and calculated structure factor respectively. was calculated using of the data. in a of structure of the TreX shows similarity to its enzyme, Pseudomonas isoamylase, with a of Å It of three the and The N-terminal (amino acids a The contains the found in of the consisting of by The of TreX on the isoamylase shows similarity in of the including the region of an region of and of and The in isoamylase and other is not found in the TreX it has two and and and we that TreX exists in two oligomeric in solution (5Park J.-T. Park H.S. Kang H.K. Hong J.S. Cha H. Woo E.-J. Kim J.W. Kim M.J. Boos W. Lee S. Park K.-H. Biocatal. Biotransform. 2008; 26: 76-85Crossref Scopus (17) Google Scholar). both of crystals and determined the structures of the dimer and tetramer. In the dimeric each is side by side with the active site of each the side The dimeric of TreX a of to of the per with and In the tetrameric two each other with a slight as a dimer of the of each subunit that are connected to the inside of the tetramer. The tetrameric of TreX of in which of the residues from in the Structure of the with the catalytic residues and residues in the GH13 are located at the of the active-site A crystal in acarbose shows an electron density at -1 to in the active the density at -1 clearly to be connected to with density at The for the subsites that of Biochem. J. 1997; PubMed Scopus Google Scholar). The glucose at the reducing of the acarbose is to be resulting in the intermediate bound to The to subsites to a similar mode to the acarbose intermediate in the structure T. M.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus (60) Google Scholar, K. T. W. J. Biochem. 2000; Google Scholar). The of the and the of the -1 is to the of the -1 The intermediate was by at a at which acarbose is by TreX. and form the -1 of the that is for the with substrate glucose a bond with The side of the three residues Asp363, and are located in to in which the of the catalytic is at a of Å to the of the -1 the -1 and and are located in the for the enzyme at of and Å in the and acarbose complex respectively. additional glucose are at subsites and with with and and are located in a to the of the and the mode of the acarbose intermediate molecule at the active the substrate is to in a a sharp at the bond the -1 and Analysis of the region an in that the region of residues at the of the substrate is only in TreX and is not found in the isoamylase pullulanase to and is in and is in a with the the configuration of the may provide a for the of the the substrate of TreX. In comparison with isoamylase and TreX is unique in higher activity on with (5Park J.-T. Park H.S. Kang H.K. Hong J.S. Cha H. Woo E.-J. Kim J.W. Kim M.J. Boos W. Lee S. Park K.-H. Biocatal. Biotransform. 2008; 26: 76-85Crossref Scopus (17) Google Scholar). the by tetrameric structure of TreX a configuration that to from other structure In the two each other that the active are to each other at a slight The region lid 1 and lid of one dimer is located at the active site of the other dimer molecule, reshaping the of the Although the is and in the the of and lid with the active site in the tetramer results in a with structural on the active 1 of the N-terminal is the to the glucose molecule at subsites and in the complex lid is to and A and The of the generated by the structural is accompanied by a of the in the active This from the active and from in the tetrameric the to be The to in the dimeric the of the of the shift of the the is on one side and to the of the molecule to form connected with an of the of the this and the geometrical of the the connected to be a for the of a substrate The region was found to a with an to the of different in enzymes of this family T. M.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus (60) Google Scholar, K. T. W. J. Biochem. 2000; Google Scholar, H. E. J. Mol. Biol. PubMed Scopus Google Scholar, H. T. J. Mol. Biol. PubMed Scopus Google Scholar). However, the in the TreX tetramer a different with a large of has upon This contains residues as the catalytic and residues and In the dimeric the side of and by to at subsites and In the the side chain of and and at and Å, resulting in from the other catalytic residues to as a It is that TreX this with an to of as and for of 1 in structural with other enzymes with α-1,4-transferase activity, we an enzymes with α-1,4-transferase activity a structural lid the as a common similar to that in the TreX tetramer To the structural lid the active site is to the α-1,4-transferase activity of and which are in the of lid 1 and lid were to Enzyme of the mutants revealed a the enzyme α-1,4-transferase activity compared with the the enzyme the of this region in the α-1,4-transferase activity In the a relatively in the α-1,6-glucosidase activity, and the an activity level to that of the of the of in the catalytic mechanism of the active site of the molecule has because it is located at a from the active site the of the substrate used in the is not to the region from the active is another activity associated with this N-terminal region of lid the in the α-1,4-transferase activity in the TreX is by the in the of this region with the substrate in the active This provides for modulation of the bifunctional activities by tetramerization of its TreX is a of the GH13 family of to which isoamylase belongs C. Protein PubMed Scopus Google Scholar, S. Biochim. Biophys. Acta. 2001; PubMed Scopus Google Scholar). TreX is of its homologs, it exhibits two the α-1,4-transferase activity and the α-1,6-glucosidase activity. In an to understand the bifunctional activities of we carried out an analysis of the active of enzymes in the GH13 family α-1,4-transferase α-1,6-glucosidase activity for which structures are For α-1,4-transferase activity, and were because represent enzymes with high activity for as TreX. In the structural an was the enzymes with α-1,4-transferase activity Protein Data Bank for for and for a structural lid region located to the active For has a at the active site with a structural lid that is formed This oligomerization is be for the enzyme to unique substrate H. T. J. Mol. Biol. PubMed Scopus Google Scholar, J.S. Cha Kim Kim T.J. H.S. M.J. Kim M.J. Lee H.S. Kim J.W. Park K.-H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H.S. Kim H.S. Kim Kim T.J. Park C. Lee H.S. Park K.-H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the structure of a in the N-terminal from the molecule a the active site that the of the substrate at the site A. C. S. W. Rice J. Mol. Biol. PubMed Scopus Google Scholar). In the the is in T. M.J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus (60) Google Scholar), the lid from the the active site upon of the substrate in the structure H. S. Yamamoto M. T. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In to the region various mutants of that region its in activity S. H. 1998; PubMed Scopus Google Scholar). The structure of the TreX tetramer shows the structural lid on the active site generated by the similar structural exists in the that the α-1,4-transferase activity may be by the oligomeric in this region a in α-1,4-transferase activity without of the α-1,6-glucosidase activity of TreX. on the structural the of the lid region is to with and the molecule H. T. J. Mol. Biol. PubMed Scopus Google Scholar, K. J. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). the of two TreX may from a glycogen substrate in the of trehalose the active site by oligomeric as in TreX is not a in In the N-terminal of the pullulanase molecule is located the active site of the other subunit in the dimeric with residues of the N-terminal with the bound to the other H. E. J. Mol. Biol. PubMed Scopus Google Scholar). The of this dimer is to only in the presence of in solution. the of the N-terminal in and substrate has in K.-H. Kim T.J. Cheong T.K. Kim J.W. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar), from T. T. S. Biosci. Biotechnol. Biochem. 2001; 65: PubMed Scopus Google Scholar), and Kim T.J. J.W. Park Lee Kim Park K.-H. Kim J.W. Biotechnol. Appl. Biochem. PubMed Scopus Google Scholar). by an oligomeric TreX the active site for modulation of the dual activities and a connected inside the tetramer molecule, a for the of a on a previous mutational study, two activities of yeast GDE were to be and located at different on a single polypeptide with the and in the and A. Yamamoto K. Tabata S. J. Biol. Chem. 2001; 276: 28824-28828Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, W. C. 1991; PubMed Scopus Google Scholar). we that α-1,4-transferase and α-1,6-glucosidase activities at the catalytic site in TreX. In this it be to GDEs in higher a similar configuration of the active In we were to the dual activities of the archaeal GDE TreX by mutational study and provided that the α-1,4-transferase activity is closely associated with the structural lid generated by This study the structural basis for the modulation of the dual activities of TreX upon the at beamlines and Pohang Accelerator Laboratory, for data and