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• W2047583394 abstract "Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1–4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N′ domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N′ domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes. Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1–4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N′ domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N′ domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes. Many organisms live in physically or geochemically extreme conditions that are detrimental to most organisms. Most eukaryotic organisms cannot tolerate temperatures higher than 50 °C, due to the sensitivity of certain cellular components. The discovery of hyperthermophilic microorganisms living near, at, and above 100 °C has revolutionized scientific thought in this area, and many scientists have become interested in such systems because they have developed specific physicochemical characteristics. Due to these particular properties, enzymes from extremophiles offer great potential for basic research and for biotechnological applications. For example, most industrial starch processes require high temperatures for liquefaction and saccharification. Therefore, there is enthusiastic interest in finding new sources of thermostable amylolytic enzymes. Recently, we reported a novel maltogenic amylase from Staphylothermus marinus (1.Li D. Park J.T. Li X. Kim S. Lee S. Shim J.H. Park S.H. Cha J. Lee B.H. Kim J.W. Park K.H. Overexpression and characterization of an extremely thermostable maltogenic amylase, with an optimal temperature of 100 degrees C, from the hyperthermophilic archaeon Staphylothermus marinus.New Biotechnol. 2010; 27: 300-307Crossref PubMed Scopus (31) Google Scholar) that was isolated from geothermal sediments from a “black smoker” on the ocean floor (2.Fiala G.S.K. Jannasch H.W. Langworthy T.A. Madon J. Staphylothermus marinus sp. nov. represents a novel genus of extremely thermophilic submarine heterotrophic archaebacteria growing up to 98 °C.Syst. Appl. Microbiol. 1986; 8: 106-113Crossref Scopus (158) Google Scholar). Maltogenic amylase is an enzyme that is widely used in the starch industry. This enzyme exhibits dual activity for α-d-1,4- and α-d-1,6-glucosidic bond cleavage, which differs from the classic α-amylases in the glycoside hydrolase 13 (GH13) 5The abbreviations used are: GH13glycoside hydrolase 13ADAN-(2-acetamido)-2-iminodiacetic acidThMAmaltogenic amylase from Thermus sp. strain IM6501NPaseneopullulanase from B. stearothermophilusCDasecyclodextrinase from alkalophilic Bacillus sp. I-5TVAI and TVAIIT. vulgaris amylase I and II, respectivelySMMAS. marinus maltogenic amylasePFTAP. furiosus thermostable amylaseCDcyclodextrinCBMcarbohydrate-binding moduler.m.s.root mean squareaaamino acidsAMPK5′-AMP-activated protein kinase. family (3.MacGregor E.A. Janecek S. Svensson B. Relationship of sequence and structure to specificity in the α-amylase family of enzymes.Biochim. Biophys. Acta. 2001; 1546: 1-20Crossref PubMed Scopus (553) Google Scholar). SMMA has an optimal temperature of 100 °C and a 109 °C melting temperature with enzymatic activity under acidic conditions (pH 3.5–5.0), which is a favorable property for industrial applications (2.Fiala G.S.K. Jannasch H.W. Langworthy T.A. Madon J. Staphylothermus marinus sp. nov. represents a novel genus of extremely thermophilic submarine heterotrophic archaebacteria growing up to 98 °C.Syst. Appl. Microbiol. 1986; 8: 106-113Crossref Scopus (158) Google Scholar, 4.Unsworth L.D. van der Oost J. Koutsopoulos S. Hyperthermophilic enzymes. Stability, activity, and implementation strategies for high temperature applications.FEBS J. 2007; 274: 4044-4056Crossref PubMed Scopus (165) Google Scholar). The conversion of starch at a high temperature and low pH offers several advantages, including higher substrate solubility, decreased viscosity, better bacterial decontamination, and increased reaction rates (4.Unsworth L.D. van der Oost J. Koutsopoulos S. Hyperthermophilic enzymes. Stability, activity, and implementation strategies for high temperature applications.FEBS J. 2007; 274: 4044-4056Crossref PubMed Scopus (165) Google Scholar, 5.Auh J.H. Chae H.Y. Kim Y.R. Shim K.H. Yoo S.H. Park K.H. Modification of rice starch by selective degradation of amylose using alkalophilic Bacillus cyclomaltodextrinase.J. Agric. Food Chem. 2006; 54: 2314-2319Crossref PubMed Scopus (34) Google Scholar, 6.Kawamura S. Kakuta Y. Tanaka I. Hikichi K. Kuhara S. Yamasaki N. Kimura M. Glycine 15 in the bend between two α-helices can explain the thermostability of DNA-binding protein HU from Bacillus stearothermophilus.Biochemistry. 1996; 35: 1195-1200Crossref PubMed Scopus (44) Google Scholar, 7.Kim Y.W. Choi J.H. Kim J.W. Park C. Kim J.W. Cha H. Lee S.B. Oh B.H. Moon T.W. Park K.H. Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance.Appl. Environ. Microbiol. 2003; 69: 4866-4874Crossref PubMed Scopus (88) Google Scholar, 8.Vieille C. Zeikus G.J. Hyperthermophilic enzymes: Sources, uses, and molecular mechanisms for thermostability.Microbiol. Mol. Biol. Rev. 2001; 65: 1-43Crossref PubMed Scopus (1652) Google Scholar). glycoside hydrolase 13 N-(2-acetamido)-2-iminodiacetic acid maltogenic amylase from Thermus sp. strain IM6501 neopullulanase from B. stearothermophilus cyclodextrinase from alkalophilic Bacillus sp. I-5 T. vulgaris amylase I and II, respectively S. marinus maltogenic amylase P. furiosus thermostable amylase cyclodextrin carbohydrate-binding module root mean square amino acids 5′-AMP-activated protein kinase. The GH13 family includes α-amylases and closely related subfamilies, such as maltogenic amylase (EC 3.2.1.133), neopullulanase (EC 3.2.1.135), Thermoactinomyces vulgaris amylase II (TVAII), and cyclomaltodextrinase (CDase; EC 3.2.1.54) (3.MacGregor E.A. Janecek S. Svensson B. Relationship of sequence and structure to specificity in the α-amylase family of enzymes.Biochim. Biophys. Acta. 2001; 1546: 1-20Crossref PubMed Scopus (553) Google Scholar). Maltogenic amylase shares catalytic characteristics with neopullulanase and CDase, which catalyze the hydrolysis of cyclodextrins (CDs), pullulan, and acarbose, and which are collectively known as CD-hydrolyzing enzymes (9.Park K.H. Kim T.J. Cheong T.K. Kim J.W. Oh B.H. Svensson B. Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family.Biochim. Biophys. Acta. 2000; 1478: 165-185Crossref PubMed Scopus (186) Google Scholar). These enzyme specificities were proposed to establish the so-called neopullulanase subfamily of the α-amylase family GH13 (10.Oslancová A. Janecek S. Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the alpha-amylase family defined by the fifth conserved sequence region.Cell Mol. Life Sci. 2002; 59: 1945-1959Crossref PubMed Scopus (81) Google Scholar), which is currently classified as the subfamily GH13_20 (11.Stam M.R. Danchin E.G. Rancurel C. Coutinho P.M. Henrissat B. Dividing the large glycoside hydrolase family 13 into subfamilies. Towards improved functional annotations of α-amylase-related proteins.Protein Eng. Des. Sel. 2006; 19: 555-562Crossref PubMed Scopus (433) Google Scholar). CD-hydrolyzing enzymes possess a common domain at the N terminus (N domain) that is involved in substrate binding through a domain-swapped homodimeric structure (12.Hondoh H. Kuriki T. Matsuura Y. Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase.J. Mol. Biol. 2003; 326: 177-188Crossref PubMed Scopus (121) Google Scholar, 13.Kamitori S. Abe A. Ohtaki A. Kaji A. Tonozuka T. Sakano Y. Crystal structures and structural comparison of Thermoactinomyces vulgaris R-47 α-amylase 1 (TVAI) at 1.6 Å resolution and α-amylase 2 (TVAII) at 2.3 Å resolution.J. Mol. Biol. 2002; 318: 443-453Crossref PubMed Scopus (53) Google Scholar, 14.Kim T.J. Kim M.J. Kim B.C. Kim J.C. Cheong T.K. Kim J.W. Park K.H. Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.Appl Environ. Microbiol. 1999; 65: 1644-1651Crossref PubMed Google Scholar, 15.Lee H.S. Kim M.S. Cho H.S. Kim J.I. Kim T.J. Choi J.H. Park C. Lee H.S. Oh B.H. Park K.H. Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other.J. Biol. Chem. 2002; 277: 21891-21897Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 16.Ohtaki A. Mizuno M. Yoshida H. Tonozuka T. Sakano Y. Kamitori S. Structure of a complex of Thermoactinomyces vulgaris R-47 α-amylase 2 with maltohexaose demonstrates the important role of aromatic residues at the reducing end of the substrate binding cleft.Carbohydr Res. 2006; 341: 1041-1046Crossref PubMed Scopus (20) Google Scholar). The narrow, deep active site generated by the N domain from another subunit in the dimer is responsible for the substrate preference, for example, for small substrates and CDs. SMMA has enzyme activity similar to the CD-hydrolyzing enzymes with broad substrate specificity. Interestingly, the primary structure analysis of SMMA revealed that most thermostable maltogenic amylases from archaea, such as S. marinus (1.Li D. Park J.T. Li X. Kim S. Lee S. Shim J.H. Park S.H. Cha J. Lee B.H. Kim J.W. Park K.H. Overexpression and characterization of an extremely thermostable maltogenic amylase, with an optimal temperature of 100 degrees C, from the hyperthermophilic archaeon Staphylothermus marinus.New Biotechnol. 2010; 27: 300-307Crossref PubMed Scopus (31) Google Scholar), Thermofilum pendens Hrk5 (17.Li X. Li D. Yin Y. Park K.H. Characterization of a recombinant amylolytic enzyme of hyperthermophilic archaeon Thermofilum pendens with extremely thermostable maltogenic amylase activity.Appl. Microbiol. Biotechnol. 2010; 85: 1821-1830Crossref PubMed Scopus (24) Google Scholar), Thermoplasma volcanium GSS1 (18.Kawashima T. Amano N. Koike H. Makino S. Higuchi S. Kawashima-Ohya Y. Watanabe K. Yamazaki M. Kanehori K. Kawamoto T. Nunoshiba T. Yamamoto Y. Aramaki H. Makino K. Suzuki M. Archaeal adaptation to higher temperatures revealed by genomic sequence of Thermoplasma volcanium.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 14257-14262Crossref PubMed Scopus (166) Google Scholar), and Pyrococcus furiosus (19.Yang S.J. Lee H.S. Park C.S. Kim Y.R. Moon T.W. Park K.H. Enzymatic analysis of an amylolytic enzyme from the hyperthermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an α-amylase and a cyclodextrin-hydrolyzing enzyme.Appl. Environ. Microbiol. 2004; 70: 5988-5995Crossref PubMed Scopus (56) Google Scholar), have a longer motif at the N-terminal region that is 220–250 amino acids long. However, other bacterial maltogenic amylases and CD-hydrolyzing enzymes have N-terminal regions with only 120–140 amino acids that in the CAZy (Carbohydrate-active Enzyme) server have been classified as the carbohydrate-binding module (CBM) family 34, based on the observation that this domain from the related α-amylase I (TVAI) in T. vulgaris binds carbohydrates (20.Abe A. Tonozuka T. Sakano Y. Kamitori S. Complex structures of Thermoactinomyces vulgaris R-47 α-amylase 1 with malto-oligosaccharides demonstrate the role of domain N acting as a starch-binding domain.J. Mol. Biol. 2004; 335: 811-822Crossref PubMed Scopus (85) Google Scholar, 21.Machovic M. Janecek S. Starch-binding domains in the post-genome era.Cell Mol. Life Sci. 2006; 63: 2710-2724Crossref PubMed Scopus (115) Google Scholar). Because it is the most thermostable maltogenic amylase yet reported, we studied the SMMA structure for detailed information about its function. In this study, we show the three-dimensional structure of the enzyme, which reveals a unique domain arrangement in the active site associated with the N-terminal region that distinguishes the archaeal maltogenic amylase from classic bacterial maltogenic amylases. The strain Escherichia coli MC1061 (F-7, araD139, Δ(ara-leu)7696, galE15, galK16, Δ(lac)X74, rpsL,(Strr), hsdR2, (rk−mk+), mcrA, and mcrB1) harboring pSMMA6xH was cultured for 20 h in 3 liters of Luria-Bertani broth. The cells were collected by centrifugation (8000 × g, 30 min), resuspended in 300 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 300 mm NaCl, and 10 mm imidazole), and sonicated. The supernatant was collected by centrifugation (10,000 × g, 30 min, 4 °C) and heated at 70 °C for 20 min to remove the thermolabile E. coli proteins. The crude enzyme was further purified using a nickel-NTA Superflow® column, as described previously (14.Kim T.J. Kim M.J. Kim B.C. Kim J.C. Cheong T.K. Kim J.W. Park K.H. Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.Appl Environ. Microbiol. 1999; 65: 1644-1651Crossref PubMed Google Scholar). The active fractions in the elution buffer were dialyzed against 50 mm Tris-HCl buffer (pH 7.5). SMMA crystallization trials were conducted using the sitting drop method at 18 °C. We mixed 1.5 μl of a 14 mg/ml SMMA solution with an equal volume of crystallization reservoir solution containing 12% polyethylene glycol (PEG) 4000, 2% isopropyl alcohol, 0.1 m ADA, pH 6.5, and 0.1 m Li2SO4. Before data collection, rhombus-type crystals were cryocooled to 95 K using a cryoprotectant consisting of mother liquor supplemented with 25% glycerol. The crystal diffracted to a resolution of 2.28 Å, and the data were collected with a 1° rotation and a total of 340 frames. Diffraction data were processed and scaled using HKL2000 (22.Otwinowski Z. Minor M. Processing of x-ray diffraction data collected in oscillation mode.Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38565) Google Scholar). The structure was determined using the molecular replacement method with the Phaser CCP4 suite (23.Collaborative Computational Project, Number 4 The CCP4 suite. Programs for protein crystallography.Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19762) Google Scholar) and the neopullulanase from Bacillus stearothermophilus (Protein Data Bank entry 1J0H) with the N domain omitted. The resulting model was refined through model rebuilding using CNS (24.Brünger A.T. Adams P.D. Rice L.M. Recent developments for the efficient crystallographic refinement of macromolecular structures.Curr. Opin. Struct. Biol. 1998; 8: 606-611Crossref PubMed Scopus (81) Google Scholar). COOT (25.Emsley P. Cowtan K. Coot. Model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23344) Google Scholar) was used for stereographic manual refinement and model building. The structure was validated with PROCHECK (26.Laskowski R.A. Moss D.S. Thornton J.M. Main-chain bond lengths and bond angles in protein structures.J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1083) Google Scholar). Structure-based sequence alignments were generated using ClustalW (27.Thompson J.D. Higgins D.G. Gibson T.J. ClustalW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55720) Google Scholar). Molecular images, including schematics and stick figures, were produced using PyMOL (28.DeLano W.L. The PyMOL Molecular Graphics System, version 1.3r1. Schrodinger, LLC, New York2010Google Scholar). The detailed statistics for data collection and refinement are listed in Table 1.TABLE 1Data collection and structure-solution parametersParametersValuesCrystal typeNativeUnit cell parameters (Å)a = 65.39, b = 117.51, c = 199.04Resolution (Å)50–2.28Space groupP212121Completeness (%)85.6 (72.9)aNumbers in parentheses are statistics from the highest resolution shell.Rsym (%)bRsym = |Iobs − Iavg|/Iobs, where Iobs is the observed individual reflection, and Iavg is the average over all symmetry equivalents.4.8 (19.8)I/σ(I)37.1 (4.9)No. of refined atoms: protein/water11,565/385Rfactor/Rfree (%)cR factor = |Fo| − |Fc‖/|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree was calculated using 5% of the data.18.7/23.7r.m.s. deviation bond length (Å)0.012r.m.s. deviation bond angle (degrees)1.116Ramachandran plot (%)Most favored region1293 (94.6%)Additionally allowed region74 (5.4%)Outlier region0 (0%)a Numbers in parentheses are statistics from the highest resolution shell.b Rsym = |Iobs − Iavg|/Iobs, where Iobs is the observed individual reflection, and Iavg is the average over all symmetry equivalents.c R factor = |Fo| − |Fc‖/|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree was calculated using 5% of the data. Open table in a new tab The SMMA and β-cyclodextrin ligand complex model was constructed by overlaying the SMMA structure onto the ThMA complex structure (Protein Data Bank entry 1GVI). The substrate was optimized manually prior to energy minimization by using the steepest descent method with an 8-Å cut-off for 300 iterations using InsightII (Accerlys, San Diego, CA). A BLAST (29.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70619) Google Scholar) search on SMMA (GenBankTM accession number ABN69720.1) showed that the encoded protein has 23–28% identity with other GH13 proteins, including cyclomaltodextrinase from Thermococcus sp. B1001 (30.Hashimoto Y. Yamamoto T. Fujiwara S. Takagi M. Imanaka T. Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.J. Bacteriol. 2001; 183: 5050-5057Crossref PubMed Scopus (60) Google Scholar), maltogenic amylase from Thermus sp. IM6501 (14.Kim T.J. Kim M.J. Kim B.C. Kim J.C. Cheong T.K. Kim J.W. Park K.H. Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.Appl Environ. Microbiol. 1999; 65: 1644-1651Crossref PubMed Google Scholar), neopullulanase from B. stearothermophilus (31.Imanaka T. Kuriki T. Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan.J. Bacteriol. 1989; 171: 369-374Crossref PubMed Google Scholar), and T. vulgaris α-amylase II (13.Kamitori S. Abe A. Ohtaki A. Kaji A. Tonozuka T. Sakano Y. Crystal structures and structural comparison of Thermoactinomyces vulgaris R-47 α-amylase 1 (TVAI) at 1.6 Å resolution and α-amylase 2 (TVAII) at 2.3 Å resolution.J. Mol. Biol. 2002; 318: 443-453Crossref PubMed Scopus (53) Google Scholar). The copper bicinchoninate method was used to measure the concentration of reducing products to determine activity and kinetic parameters of SMMA (15.Lee H.S. Kim M.S. Cho H.S. Kim J.I. Kim T.J. Choi J.H. Park C. Lee H.S. Oh B.H. Park K.H. Cyclomaltodextrinase, neopullulanase, and maltogenic amylase are nearly indistinguishable from each other.J. Biol. Chem. 2002; 277: 21891-21897Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 32.Fox J.D. Robyt J.F. Miniaturization of three carbohydrate analyses using a microsample plate reader.Anal. Biochem. 1991; 195: 93-96Crossref PubMed Scopus (361) Google Scholar). For thin layer chromatography (TLC) analysis, the reaction products were spotted onto Whatman K5F silica gel plates (Whatman plc, Maidstone, UK) and developed using isopropyl alcohol/ethyl acetate/water (3:1:1, v/v/v) as the solvent system. Proximity to the end of a secondary structure element is defined as being within 4 amino acids for helix and within 1 amino acid for strand, where the termini are assigned from the Protein Data Bank (33.Greaves R.B. Warwicker J. Stability and solubility of proteins from extremophiles.Biochem. Biophys. Res. Commun. 2009; 380: 581-585Crossref PubMed Scopus (14) Google Scholar). Structures used in the analysis are Protein Data Bank entries 1GVI (ThMA), 1J0H (NPase), 1JI2 (TVAII), and 1EA9 (CDase). Residues in the exposed surfaces were identified using the Areaimol program (34.Lee B. Richards F.M. The interpretation of protein structures. Estimation of static accessibility.J. Mol. Biol. 1971; 55: 379-400Crossref PubMed Scopus (5341) Google Scholar). The crystal structure revealed that SMMA comprises four domains: the N, catalytic, and C domains, which are observed in most CD-hydrolyzing enzymes, and an additional novel N-terminal domain, the N′ domain, which was first observed in this study (Fig. 1a). Initially, the structure was determined and refined to a 2.28 Å resolution using molecular replacement, with the catalytic and C domains of neopullulanase from B. stearothermophilus (Protein Data Bank entry 1J0H) as the template structure. An attempt at molecular replacement with the entire three-domain region failed, which may have been due to the significantly altered orientation and geometry of the SMMA N domain (r.m.s. deviation of 2.3 Å for 463 Cα atoms). During manual model building for the N-terminal region, the electron density of the region showed two vague, separate “blobs,” which allowed the detection of the N (aa 116–219) and N′ (aa 1–115) domains of SMMA. The SMMA catalytic domain displays a conserved (β/α) 8-barrel fold with a distinct loop (aa 342–397) protruding from the barrel. Most CD-hydrolyzing enzymes have a protruding loop located near the active site, which is called the B domain and forms a portion of the substrate binding groove for subsites −2, −3, or −4. However, SMMA has a much longer insertion of aa 342–397 in this region, creating a larger domain at the entrance of the groove (Fig. 1b). In this groove, Tyr-389, which is in the middle of a helix in aa 385–391, forms an entrance gate with Tyr-257. Because SMMA and other CD-hydrolyzing enzymes from archaea have this insertion, the protruding region might serve as a signature that is unique to maltogenic amylases from archaea. Another sequence-structural feature is the presence of a glycine in the i + 4 position after the catalytic nucleophile Asp-442 (Fig. 2) because typical maltogenic amylases (i.e. members of the neopullulanase subfamily) have a glutamate in that position as a part of the four-residue signature VANE (10.Oslancová A. Janecek S. Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the alpha-amylase family defined by the fifth conserved sequence region.Cell Mol. Life Sci. 2002; 59: 1945-1959Crossref PubMed Scopus (81) Google Scholar).FIGURE 2Sequence alignment for SMMA, PFTA, ThMA, NPase, TVAII, and CDase. The sequences of six CD-hydrolyzing enzymes, two hyperthermophilic archaic maltogenic amylases, and four conventional bacterial CD-hydrolyzing enzymes were aligned using ClustalW (27.Thompson J.D. Higgins D.G. Gibson T.J. ClustalW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55720) Google Scholar). The novel SMMA N′ domain is indicated with a blue dotted line. The helix-loop-helix regions observed in the structure are indicated with orange dotted lines. The β-7-loop 7-β-8 region protruding from the N′ domain is indicated with a pink dotted line. The three conserved catalytic residues in the GH13 family are indicated with black asterisks. Red, blue, and green highlighting indicates the residues that are conserved in 100, 75, and 50% of the proteins, respectively. The accession numbers for these sequences are ABN69720.1 (SMMA), AAL82063.1 (PFTA from Pyrococcus furiosus), AAC15072.1 (ThMA from Thermus sp. strain IM6501), AAA22622.1 (NPase from B. stearothermophilus), BAA02473.1 (TVAII), and AAA92925.1 (CDase from alkalophilic Bacillus sp. I-5).View Large Image Figure ViewerDownload Hi-res image Download (PPT) A Dali search (35.Holm L. Kääriäinen S. Rosenström P. Schenkel A. Searching protein structure databases with DaliLite version 3.Bioinformatics. 2008; 24: 2780-2781Crossref PubMed Scopus (845) Google Scholar) using the SMMA structure identified the neopullulanase from B. stearothermophilus (29% identity) as its closest structural homologue, with a 2.6 Å r.m.s. deviation (536 Cα atoms). The α amylase II (TVAII) from T. vulgaris R-47 (30% identity) was identified as the second closest homologue with a 2.6 Å r.m.s. deviation (537 Cα atoms). The loop regions comprising aa 236–264 and 653–689 had high B-factors (61.3 average) at the surface, and the electron density map for the loop of aa 671–677 in the C domain was too weak to build a model, which suggests that it may be flexible. SMMA forms a homodimer via an interaction between the adjacent, novel N′ domains, which have a 2-fold axis perpendicular to the arc shape of the β-strands' interface (Fig. 1c). Each monomer is primarily associated through hydrophobic interactions at the center of the region of aa 5–19 (Ile-5 and -19 from one molecule against Ile-9 from the other). This interaction is supplemented by salt bridges (Arg-181/Asp-422 and Arg-50/Glu-198) at both ends of the strands, which yield a 2140.7 Å2 interface (Fig. 1d). Most CD-hydrolyzing enzymes form dimers with the N domain intertwined. However, the SMMA dimer configuration is different from previously reported CD-hydrolyzing enzymes, in that the dimer is arranged with adjacent monomers and an interface unrelated to the active sites. The long N-terminal region of SMMA includes two repeated motifs, the N and N′ domains, with a β-sandwich fold (6.2 Å r.m.s. deviation for 12 Cα atoms) (Fig. 3a). A structural homology search for the truncated N′ domain generated the β-subunit of the 5′-AMP-activated protein kinase (AMPK) with a 1.9 Å r.m.s. deviation over 80 residues (Z score, 10.2) using the Dali and 1.71 Å over 80 residues (Z score, 6.2) using the SSM server (36.Krissinel E. Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2256-2268Crossref PubMed Scopus (3160) Google Scholar). The AMPK β subunit, which is known to bind glycogen, belongs to CBM48 (37.Polekhina G. Gupta A. van Denderen B.J. Feil S.C. Kemp B.E. Stapleton D. Parker M.W. Structural basis for glycogen recognition by AMP-activated protein kinase.Structure. 2005; 13: 1453-1462Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Currently, the CBM48 family has more than 3000 entries in the CAZy database, and 15 entries have structural information available (38.Cantarel B.L. Coutinho P.M. Rancurel C. Bernard T. Lombard V. Henrissat B. The Carbohydrate-active enzymes database (CAZy). An expert resource for glycogenomics.Nucleic Acids Res. 2009; 37: D233-D238Crossref PubMed Scopus (4129) Google Scholar). Despite the low overall sequence similarity, all of the structures superimposed well onto the SMMA N′ domain and share 6–8 β-strands from the β-sandwich fold, except for the long extended loop of aa 88–110 between the 7th and 8th β strands in SMMA (Fig. 3b). Eight of 15 structures have protruding loops that correspond to the loop of aa 88–110 in SMMA, but the SMMA β-strand loop is much longer (8.2 Å) than the other corresponding loo" @default.
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• W2047583394 date "2012-03-01" @default.
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• W2047583394 title "Association of Novel Domain in Active Site of Archaic Hyperthermophilic Maltogenic Amylase from Staphylothermus marinus" @default.
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