Crystal structure of amyb, an alpha amylase from halothermothrix orenii, and comparison with its homologs

CRYSTAL STRUCTURE OF AMYB, AN αAMYLASE FROM HALOTHERMOTHRIX ORENII, AND COMPARISON WITH ITS HOMOLOGS Tien Chye Tan Submitted 14 April 2007 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 PUBLICATIONS Paper I Tan, T.C., Yien, Y.Y., Patel, B.K., Mijts, B.N., and Swaminathan, K. (2003). Crystallization of a novel alpha-amylase, AmyB, from the thermophilic halophile Halothermothrix orenii. Acta Crystallografica sect. D 59, 2257-2258. Paper II Huynh, F., Tan, T.C., Swaminathan, K., and Patel, B.K. (2005). Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii. Acta Crystallographica sect. F 61, 116-117. I ACKNOWLEDGEMENTS In my list of acknowledgements, there are people with specific contributions and also people who have helped me in many small but important ways that are impossible to list. The following list is by no means complete nor are the contributions of those listed limited to what is listed. It is just a weak attempt to thank everyone for their help and kindness shown to me during my time here. Dr Victor Wong and Dr Kunchithapadam Swaminathan for their patience and guidance. Dr Wong for getting me started in research and for believing in me. Dr Swami for burning the midnight oil to help me get my thesis out on time. My collaborator Dr Bharat Patel for spending hours educating me on his field. Dr Jayaraman Sivaraman for putting up with my endless list of questions. Thanks also go out to my ‘editor’ Maykalavaane d/o Narayanan for helping make sense of my disjointed thoughts and my dyslexic spelling. My lab mates, members of Structural Biology Lab for the mayhem and chaos to spice up our stay here. Not forgetting the department staff who definitely have help a lot in their own way. Dr Christina Divne for supervision on additional experimental results presented in the revised version of the thesis, and advice on revision of the thesis. And for pulling my brain out of the gutter and getting it to stay focused. Prof Birte Svensson and Dr Karen Marie Jakobsen at BioCentrum-DTU (Lyngby, Denmark) for supplying the acarbose inhibitor, which was put to good use. Of course there is my mom but that would take up another thesis on its own so I shall just say “THANKS MOM”. II Table of Contents Publications I Acknowledgements II Table of Contents III List of Tables VII List of Figures VIII List of abbreviations used Summary 1.1 1.2 1.3 1.4 X XIII INTRODUCTION Extremophiles 1.1.1 Adaptation to high salinity 1.1.2 Thermal adaptation 1.1.3 Stabilization mechanisms Classification of carbohydrate-active enzymes 1.2.1 Carbohydrates as enzyme substrates 1.2.2 Carbohydrate-active enzymes are classified in CAZy 1.2.3 Reaction mechanisms of glycoside hydrolases 1.2.4 Modes of action in GH enzymes 11 1.2.5 The TIM barrel is a recurrent fold in GH enzymes 13 1.2.6 Linking CBMs to catalytic domains 15 Starch and starch-processing enzymes 17 1.3.1 Starch – the enzyme substrate 17 1.3.2 Starch-processing enzymes 20 1.3.3 The α-amylase superfamily 23 1.3.4 Starch-binding CBMs 27 The bacterium Halothermothrix orenii and its α-amylases 28 1.4.1 Halothermothrix orenii 28 III 1.4.2 Halothermothrix orenii produces two α-amylases 28 1.4.3 Biochemical characteristics of AmyA and AmyB 30 1.4.4 Thermal inactivation studies on AmyA and AmyB 31 1.4.5 Salt dependence of AmyA and AmyB 32 1.4.6 Content of charged amino acids in AmyA and AmyB 33 1.4.7 Crystal structure of AmyA 33 1.5 Applications 34 1.6 Scope of the thesis 34 2.1 2.2 2.3 MATERIALS AND METHODS 35 PCR and cloning 35 2.1.1 Construct design and PCR 35 2.1.2 Preparation of competent cells 37 2.1.3 Cloning and transformation 38 2.1.4 Preparation of the expression vector 38 Protein expression and purification 39 2.2.1 Protein expression 39 2.2.2 Protein purification 40 2.2.3 Enzyme purity 42 2.2.4 Amylase activity assay 42 2.2.5 Starch binding 43 2.2.6 Protein stability 43 Crystallographic analysis 44 2.3.1 Initial crystallization screening 44 2.3.2 Optimization of crystallization conditions and ligand soaks 45 2.3.3 X-ray diffraction data collection 45 2.3.4 Structure determination and refinement 46 2.3.5 Various analyses 48 IV RESULTS 50 3.1 Analysis of the lipoprotein signal sequence 50 3.2 Cloning and protein production 51 3.2.1 Cloning of ΔAmyB 51 3.2.2 Purification of AmyB and ΔAmyB 51 3.2.3 Starch degradation studies on AmyB and ΔAmyB 53 3.2.4 Starch binding studies on AmyB and ΔAmyB 54 3.2.5 Stability analysis of AmyB and ΔAmyB 56 3.3 Protein crystallization and optimization 58 3.4 Data collection and processing 60 3.4.1 AmyB crystal form I 60 3.4.2 AmyB crystal form II 60 3.4.3 AmyB crystal form III 61 3.4.4 AmyB crystal form IV 62 Structure determination, model building and refinement 64 3.5.1 Original AmyB structure 64 3.5.2 Acarbose complex 67 3.5.3 Maltoheptaose/cyclodextrin complex 67 The crystal structure of AmyB 68 3.6.1 Quality of the final models 68 3.6.2 Domains A and B – the catalytic module 71 3.6.3 Domain C 72 3.6.4 Domain N 74 Binding of oligosaccharides to AmyB 82 3.7.1 Binding of acarbose-derived oligosaccharide 82 3.7.2 Binding of maltoheptaose and α-cyclodextrin 90 3.7.3 The active site in AmyB 92 3.5 3.6 3.7 V 4.1 4.2 4.3 DISCUSSION 95 The natural substrate 95 4.1.1 Natural habitat of Halothermothrix orenii 95 4.1.2 Possible natural substrates for H. orenii AmyB 95 Stability and adaptation 97 4.2.1 Influence of negatively charged surfaces 97 4.2.2 Influence of the cation triad 99 4.2.3 Influence of methionine content 100 4.2.4 Thermal stability as a function of salt concentration and pH 100 AmyB represents a unique member of the α-amylase family 101 4.3.1 AmyB is a membrane-bound enzyme 101 4.3.2 Role of the N domain 103 4.3.3 AmyB is unique compared with AmyA and other α-amylases 104 CONCLUSIONS 106 REFERENCES 108 Paper I Paper II VI LIST OF TABLES Table 1.1. Categories of halophiles Table 1.2. Clans and folds of glycoside hydrolases Table 1.3. CBM fold families Table 1.4. Types of carbohydrate binding platforms Table 1.5. Characteristics of exoamylases Table 3.1. Statistics for data collection. Table 3.2. Statistics for crystallographic refinement. Table 3.3. Interface parameter analysis for domain A/N association. Table 3.4. Mapping of sugar residues of acarbose-derived oligosaccharides to the active-site subsites of the α-amylases BA2, AmyB and BHA. Table 3.5. Interactions with a nonasaccharide in the A-B groove of AmyBACR. Table 3.6. Interactions with acarbose in the N-C groove of AmyBACR. Table 3.7. Interactions with α-D-glucose in the B1 and B2 sites of AmyBACR. Table 3.8. Interactions with maltotetraose in the A-B groove of AmyBMAL7-ACX. Table 3.9. Interactions with maltotetraose in the N-C groove of AmyBMAL7-ACX. VII LIST OF FIGURES Figure 1.1. Chair representation of cellobiose. Figure 1.2. Reaction mechanisms for glycoside hydrolases. Figure 1.3. Active-site topologies of glycoside hydrolases. Figure 1.4. GH clans with the TIM-barrel fold. Figure 1.5. Sugar-binding platforms in CBMs. Figure 1.6. Structure of starch components. Figure 1.7. Helical structure of V- and A-amylose. Figure 1.8. Enzymes involved in starch processing. Figure 1.9. The domain-organization of α-amylases. Figure 1.10. The active site in Bacillus circulans strain 251 CGTase. Figure 1.11. The structure of a lipoprotein secretion-signal peptide. Figure 2.1. Schematic representation of AmyB constructs. Figure 2.2. Schematic representation of the amyB-containing pTHAB template. Figure 3.1. Lipoprotein signal peptide in AmyB. Figure 3.2. Analysis of protein purity by SDS-PAGE. Figure 3.3. Gel-filtration chromatogram for AmyB (B2) and ΔAmyB (B3). Figure 3.4. Analysis of protein purity by SDS-PAGE. Figure 3.5. Rate of starch degradation by AmyB and ΔAmyB. Figure 3.6. Binding of AmyB and ΔAmyB to raw starch as a function of [NaCl]. Figure 3.7. Tm values for AmyB and ΔAmyB as a function of NaCl concentration at different pH values. Figure 3.8. Tm values for AmyB and ΔAmyB as a function of pH at different NaCl concentrations. Figure 3.9. Morphology of slow-growing, non-optimized AmyB crystal forms I-III. Figure 3.10. Morphology of AmyB crystal form IV. Figure 3.11. Diffraction pattern of the AmyBACR crystal. Figure 3.12. Crystal packing in the C2 unit cell of the AmyB-III crystal form. VIII Figure 3.13. Ramachandran analysis of the AmyB models. Figure 3.14. Representative electron density for AmyB models. Figure 3.15. Overall structure of AmyB. Figure 3.16. Topology diagram for the catalytic A/B domain in H. orenii AmyB. Figure 3.17. Topology diagram of the AmyB-C domain. Figure 3.18. Overall fold of the AmyB-N domain. Figure 3.19. Structural superposition of H. orenii AmyB-N with P. syringae CopC. Figure 3.20. 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Zhang, L., Koay, M., Maher, M.J., Xiao, Z. & Wedd, A.G. (2006). Intermolecular transfer of copper ions from the Copc protein of Pseudomonas syringae. Crystal structures of fully loaded Cu(I)Cu(II) Forms. J. Am. Chem. Soc. 128, 5834. 123 crystallization papers Acta Crystallographica Section D Biological Crystallography Crystallization of a novel a-amylase, AmyB, from the thermophilic halophile Halothermothrix orenii ISSN 0907-4449 Tien-Chye Tan,a Yvette Y. Yien,a Bharat K. C. Patel,b Benjamin N. Mijtsb and Kunchithapadam Swaminathana,c* a Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore, bMicrobial Discovery Research Unit, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, Queensland 4111, Australia, and c Institute of Molecular and Cell Biology, 30 Medical Drive, National University of Singapore, Singapore 117609, Singapore Correspondence e-mail: dbsks@nus.edu.sg # 2003 International Union of Crystallography Printed in Denmark ± all rights reserved Acta Cryst. (2003). D59, 2257±2258 This is a report on the structure determination of AmyB, the second -amylase from Halothermothrix orenii, by X-ray crystallography. This bacterium was isolated from saltpans where conditions consisted of both high temperatures and high NaCl content. AmyB is a 599-residue protein which is stable and signi®cantly active at 358 K in starch solution containing up to 10%(w/v) NaCl. The puri®ed recombinant AmyB protein crystallizes in the monoclinic space Ê, group C2, with unit-cell parameters a = 225.85, b = 77.16, c = 50.13 A   = 99.32 , using the hanging-drop vapour-diffusion method. The Ê. crystal diffracts X-rays to a resolution limit of 1.97 A 1. Introduction Thermostable enzymes have enormous commercial potential in high-temperature industrial processes. Amylases possessing such characteristics are extremely important enzymes in the starch-hydrolysis process and have been well characterized in terms of their biochemistry and structure. To date, most of the starch-degrading enzymes studied have been from thermophilic and hyperthermophilic prokaryotes, with much less research being devoted to enzymes from thermophilic halophiles. The amylases from moderate and extreme halophiles that have been studied thus far are either active at low salt levels (halotolerant) or are inactivated at low salt levels (extremely halophilic) (Good & Hartman, 1970; Kobayashi et al., 1992; Coronado et al., 2000). None are active and stable at high temperatures (358 K and above). Biochemical characterizations of the two -amylases, AmyA (Li et al., 2002) and AmyB (this study), from the anaerobic, thermophilic (growth at temperature above 358 K) and moderately halophilic (optimum NaCl requirement of 10%) bacterium Halothermothrix orenii have been reported (Mijts & Patel, 2002). The signal peptides found at the N-terminus of both enzymes suggest that they are secreted enzymes which are stable under these extreme conditions without protection for the bacteria. Sequence alignment with other members of the -amylase family con®rms the presence of the four conserved regions of the -amylase family in both enzymes. However, AmyA and AmyB only show 23% identity and phylogenetic analysis (Fig. 1) shows that the enzymes are distinct from each other even though they are isolated from the same bacterium. This has raised important questions about the origin and evolution of the genes and their adaptation to Received 15 June 2003 Accepted 25 August 2003 the dual extreme environmental conditions. By solving the structures of both these amylases (the crystal structure of AmyA is being re®ned) and comparing them with other amylases, we hope to identify the structural features that confer functional properties and stability under such extreme conditions upon them. 2. Materials and methods 2.1. Expression and purification of recombinant AmyB The AmyB gene was cloned into the pTrcHisB vector (Invitrogen) and the protein was expressed with an N-terminal hexahistidine tag in Escherichia coli strain TOP10 cells (Invitrogen; Mijts & Patel, 2002). A single colony of TOP10 cells containing the pTrcHisAmyB construct was inoculated into 30 ml of LB-Amp medium (100 mg mlÀ1 ampicillin) and the cells were grown at 310 K for 16 h. The 30 ml culture was subsequently used to inoculate l of LB-Amp medium and the cells were grown to an OD600 of 0.6. The enzyme was induced for h by adding IPTG to a ®nal concentration of mM. The cells were pelleted by centrifugation at 7000g, resuspended in 60 ml buffer (50 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole pH 8.0) and lysed by sonication (Vibra-cell 100). Cell debris was removed from the lysate as a pellet by centrifugation at 10 000g for 30 and the supernatant was used for enzyme puri®cation. The heat-treatment method that we had used previously for purifying AmyA (Mijts & Patel, 2002) was not successful in the case of AmyB. The clari®ed supernatant was puri®ed by using Ni±NTA agarose af®nity column chromatography. For this, 20 ml of the supernatant was added to ml of Ni±NTA agarose resin (Qiagen), shaken gently for 60 at Tan et al.  AmyB 2257 crystallization papers Table Diffraction data statistics of an AmyB crystal. Values in parentheses refer to the highest resolution shell Ê ). (2.04±1.97 A Synchrotron-radiation source Ê) Wavelength (A No. of imaging plates Ê , ) Unit-cell parameters (A Space group Mosaicity of crystal ( ) Ê) Resolution range (A Total No. of re¯ections No. of unique re¯ections Redundancy Completeness (%) I/'(I) Rsym² (%) ² Rsym = Figure Phylogenetic link between AmyA and AmyB. a mixture of paratone-N and mineral oil in a 1:1 ratio and was ¯ash-cooled in liquid nitrogen. X-ray diffraction data were collected at the Advanced Photon Source (Argonne, USA) beamline 19BM with an SBC1 CCD detector. Data were collected at 100 K and were indexed, integrated and scaled using HKL2000 (Otwinowski & Minor, 1997). Figure Crystals of AmyB from H. orenii with maximum dimensions of 0.25  0.18  0.04 mm. 277 K, loaded onto a column and washed twice with 10 ml wash buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole pH 8.0). AmyB was then eluted using elution buffer (50 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole pH 8.0). Protein purity was con®rmed by SDS±PAGE analysis before pooling and concentration using Centriprep and Centricon YM-10 devices (Millipore). 2.2. Crystallization and diffraction data collection The protein was crystallized using Crystal Screen II (Hampton Research) by the sitting-drop vapour-diffusion method, in which ml of AmyB (50 mg mlÀ1) was mixed with ml of the reservoir solution and equilibrated against ml of the reservoir at 296 K. A crystal with dimensions of 0.25  0.18  0.04 mm (Fig. 2) was transferred into 2258 Tan et al.  AmyB 3. Results and discussion Puri®cation of the AmyB protein was relatively easy because of its high expression level and its halophilic characteristics. This allowed the use of a high salt concentration for the af®nity-puri®cation step which helped to reduce the non-speci®c binding of other proteins. Additional puri®cation of the protein was not necessary. From the crystal screen, three crystals were observed after four months in a condition containing 30% PEG MME 5000, 0.1 M MES pH 6.5, 0.2 M ammonium sulfate. The crystal belongs to space group Ê . The unitC2 and diffracted X-rays to 1.97 A cell parameters are a = 225.85, b = 77.16, Ê ,  = 99.32 . The Matthews c = 50.13 A coef®cient (Matthews, 1968) is calculated to Ê DaÀ1, leading to one monomer be 2.98 A molecule (MW = 72.3 kDa) in the asymmetric unit and a solvent content of 58.4%. Ê, Even though the crystal diffracted to 1.6 A the data statistics for high-resolution shells € € j i APS, USA (beamline 19BM) 0.978 480 a = 225.85, b = 77.16, c = 50.13,  = 99.32 C2 0.86 50±1.97 272674 60572 4.5 97.7 (96.5) 10.6 (1.6) 0.101 (0.320) € jhIi i À Ii ja i Ii . Ê were not acceptable. A beyond 1.97 A summary of crystallographic and datacollection statistics is reported in Table 1. We are currently in the process of solving the structure using the molecular-replacement method (using PDB entry 1vjs as the search model). We hope to compare the structures of AmyB, AmyA and other members of the amylase family that are known to be either thermophilic or halophilic in order to understand the basis of their stability, structural characteristics and functional properties under thermohalophilic conditions. Funding from the Grif®th University Grants Scheme to BKCP is gratefully acknowledged. BNM was a recipient of the Australian Commonwealth Postgraduate Award. References Coronado, M., Vargas, C., Hofemeister, J., Ventosaa, A. & Nieto, J. J. (2000). FEMS Microbiol. Lett. 183, 67±71. Good, W. A. & Hartman, P. A. (1970). J. Bacteriol. 104, 601±603. Kobayashi, T., Kanai, H., Hayashi, T., Akiba, T., Akaboshi, R. & Horikoshi, K. (1992). J. Bacteriol. 174, 3439±3444. Li, N., Patel, B. K., Mijts, B. N. & Swaminathan, K. (2002). Acta Cryst. D58, 2125±2126. Matthews, B. W. (1968). J. Mol. Biol. 33, 491±497. Mijts, B. N. & Patel, B. K. C. (2002). Microbiology, 148, 2343±2349. Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307±326. Acta Cryst. (2003). D59, 2257±2258 crystallization communications Acta Crystallographica Section F Structural Biology and Crystallization Communications ISSN 1744-3091 Frederick Huynh,a,b Tien-Chye Tan,c Kunchithapadam Swaminathanc,d and Bharat K. C. Patela,b* a Microbial Gene Research and Resources Facility, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, Queensland 4111, Australia, bEskitis Institute, Griffith University, Brisbane, Queensland 4111, Australia, cDepartment of Biological Sciences, National University of Singapore, Singapore 117543, Singapore, and d Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 118673, Singapore Correspondence e-mail: b.patel@griffith.edu.au Received 25 October 2004 Accepted 25 November 2004 Online 24 December 2004 Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii This is the ®rst report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the ®rst study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The puri®ed recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, Ê , = 103.16 , using the hanging-drop vapour-diffusion b = 47.9, c = 72.3 A Ê . Heavy-metal method. The crystal diffracts X-rays to a resolution limit of 3.01 A and halide-soaking trials are currently in progress to solve the structure. 1. Introduction Halothermothrix orenii is a unique anaerobic bacterium that grows optimally at 333 K in the presence of 10% NaCl. This combination of high temperature and high ionic strength represents one of the most extreme conditions recorded for the growth of any organism thus far (Cayol et al., 1994). These extreme growth characteristics not only make H. orenii a potentially important source for commercially useful enzymes, but also for evolutionary studies and the study of fundamental aspects of protein structure and function. We have previously reported the characteristics and crystallization of two -amylases, AmyA and AmyB (Li et al., 2002; Tan et al., 2003). Our previous studies of a random genomic DNA-sequence analysis of H. orenii identi®ed an ORF, designated spsA, with signi®cant homology to cyanobacterial sucrose phosphate synthase (SPS; EC 2.4.1.14; Mijts & Patel, 2001), an enzyme that is only ubiquitous in photosynthetic organisms, including plants and cyanobacteria. SPS, together with sucrose synthase (SS) and sucrose phosphate phosphorylase (SPP), is involved in sucrose synthesis and belongs to a group of enzymes that are collectively known as sucrose-biosynthesis proteins (SBPs). It is thought that plant SPS plays a role in osmotic regulation of intracellular turgor pressure in eukaryotes (Hite et al., 1993). To the best of our knowledge, there have been no other reports of crystal structures of SBPs, with the exception of an unreleased X-ray structure of an SPP from Synechocystis sp. PCC6803 (PDB code 1s2o). As part of our ongoing studies on H. orenii, we have overexpressed and puri®ed the spsA gene product of H. orenii. The biochemical characterization of this puri®ed recombinant spsA con®rmed its enzymatic activity, but showed signi®cant differences from the previously reported cyanobacterial and plant SPSs (Huynh, 2004). Recombinant spsA is a 61.1 kDa protein that has an optimal temperature for activity at 323±328 K and exhibits cross-reactivity with polyclonal antibodies raised against plant SPSs (AgriSera, Sweden). Like its cyanobacterial prokaryotic counterparts, the recombinant enzyme can also accept ADP-glucose and GDP-glucose as the glycosyl donor in place of UDP-glucose. By crystallizing and solving its structure, we hope to gain an insight into the structural and functional characteristics of SPS. 2. Materials and methods 2.1. Expression and purification of recombinant SPS # 2005 International Union of Crystallography All rights reserved 116 doi:10.1107/S174430910403091X The spsA gene was ampli®ed by PCR using primers incorporating BamHI and KpnI restriction-enzyme sites at the 5H and 3H ends, Acta Cryst. (2005). F61, 116±117 crystallization communications Table Diffraction data statistics. Ê ). Values in parentheses refer to the highest resolution shell (3.12±3.01 A Radiation source Rigaku RU-H3R generator and R-AXIS IV++ detector 1.5418 360 0±360 a = 154.2, b = 47.9, c = 72.3, = 103.16 C2 0.86 50±3.01 49,015 17882 2.7 (2.2) 89.7 (80.6) 0.054 (0.130) Ê) Wavelength (A No. imaging frames Crystal oscillation range ( ) Ê , ) Unit-cell parameters (A Space group Mosaicity of crystal ( ) Ê) Resolution range (A Total No. re¯ections No. unique re¯ections Redundancy Completeness (%) Rsym² (%) Figure Plate-like crystals of H. orenii SPS. The typical dimensions of diffraction-quality crystals are approximately 0.5  0.35 mm. respectively. The resulting PCR fragment was digested using these restriction enzymes, ligated into the pTrcHisA expression vector (Invitrogen) encoding an N-terminal hexahistidine fusion peptide and transformed into TransforMax Escherichia coli cells (Epicentre). Bacterial clones were cultivated in l LB medium supplemented with 50 mg mlÀ1 ampicillin at 310 K to an OD600 of 0.6. Recombinant protein expression was induced for h with IPTG to a ®nal concentration of mM. The cells were harvested by centrifugation at 5000g and resuspended in 20 ml 20 mM Tris±HCl pH 8.0, 500 mM NaCl, mg mlÀ1 lysozyme, 10 U mlÀ1 DNase I, 0.1 mg mlÀ1 RNase A, mM PMSF and one Complete EDTA-free protease-inhibitor cocktail tablet (Roche). The mixture was incubated at 310 K for 20 and sonicated on ice. Final cell lysis was achieved by three rapid freeze±thaw cycles in liquid nitrogen. Cell lysates were cleared by centrifugation at 4000 rev minÀ1 (Sigma 4K-15) for 30 and the soluble fraction was collected. HisLink Protein Puri®cation Resin (Promega) was added to the cleared cell lysate and incubated for h at 277 K with gentle agitation. The mixture was transferred to a chromatography column and washed with at least 50 column volumes of 20 mM Tris±HCl pH 7.5, 500 mM NaCl, 10 mM imidazole. Recombinant SPS was eluted with 20 mM Tris±HCl pH 7.5, 100 mM NaCl, 500 mM imidazole. Simultaneous removal of imidazole and protein concentration was achieved by several rounds of dia®ltration using an Amicon Ultra-15 Centrifugal Filter device (Millipore) with 20 mM Tris±HCl pH 7.5, 100 mM NaCl to a ®nal concentration of 10 mg mlÀ1 (Bradford method). 2.2. Crystallization Initial crystallization conditions were screened by the hangingdrop vapour-diffusion technique using the JBScreen Mixed crystallography reagent kit (Jena Bioscience). ml SPS solution was mixed with ml reservoir solution and equilibrated against 0.75 ml reservoir solution at 293 K. Single crystals appeared after d in a solution containing 16% PEG 4000, 0.1 M Tris±HCl pH 8.5, 0.2 M magnesium chloride. The condition was further optimized and crystals were obtained reproducibly with 15% PEG 3350, 0.1 M Tris±HCl pH 8.0, 0.2 M magnesium chloride with maximum dimensions of 0.5  0.35 mm after d (Fig. 1). 2.3. Data collection and analysis Crystals were picked up from the crystallization drop using nylon loops and ¯ash-cooled in liquid nitrogen. X-ray diffraction data were Acta Cryst. (2005). F61, 116±117 ² Rsym = P P j i jhIi i À Ii = P Ii . collected using an in-house Rigaku RU-H3R generator and Rigaku R-AXIS IV++ detector. All data were indexed, integrated and scaled using the programs DENZO and SCALEPACK (Otwinowski & Minor, 1997). 3. Results and discussion The spsA gene from H. orenii was successfully cloned and overexpressed and the recombinant SPS protein was puri®ed. The enzyme was con®rmed to be active at elevated temperatures and exists as a Ê and belong to monomer. The SPS crystals diffract X-rays to 3.01 A the monoclinic space group C2, with unit-cell parameters a = 154.2, Ê , = 103.16 . Crystal parameters and crystallob = 47.9, c = 72.3 A graphic data statistics are summarized in Table 1. The Matthews Ê DaÀ1 for this crystal coef®cient (VM; Matthews, 1968) of 2.13 A corresponds to an estimated solvent content of 42.2% and a monomer in the asymmetric unit. No other protein structures with sequence identity greater than 30% to SPS have been reported to date. As a consequence, molecular-replacement modelling is not an option that we can use to solve the structure and therefore attempts at heavy-atom screening for MAD analysis are under way. In addition, we hope to further optimize the cryoprotectant to reduce the relatively poor crystal mosaicity. The structure, once solved, will establish the structural fold for this family of enzymes and provide further insight into the molecular mechanism of thermostability in sucrose phosphate synthase. References Cayol, J. L., Ollivier, B., Patel, B. K., Prensier, G., Guezennec, J. & Garcia, J. L. (1994). Int. J. Syst. Bacteriol. 44, 534±540. Hite, D., Outlaw, W. H. Jr & Tarczynski, M. C. (1993). Plant Physiol. 101, 1217± 1221. Huynh, F. (2004). BSc (Hons) thesis. Grif®th University, Brisbane, Australia. Li, N., Patel, B. K., Mijts, B. N. & Swaminathan, K. (2002). Acta Cryst. D58, 2125±2126. Matthews, B. W. (1968). J. Mol. Biol. 33, 491±497. Mijts, B. N. & Patel, B. K. (2001). Extremophiles, 5, 61±69. Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307±326. Tan, T. C., Yien, Y. Y., Patel, B. K., Mijts, B. N. & Swaminathan, K. (2003). Acta Cryst. D59, 2257±2258. Huynh et al.  Sucrose phosphate synthase 117 [...]... the pyranose ring is shown for the reducing-end glucosyl moiety of maltose The α-1,4 and α-1,6 glycosidic bonds are indicated Amylose is linear and unbranched whereas amylopectin is branched with α-1,6 glycosidic bonds at the branch points The relative ratios of amylose and amylopectin vary with typical values of 15-25% and 75-85%, respectively The degree of polymerization (DP) of the nonbranched,... DP of amylopectin can be as high as 2,000,000 glucose units with roughly 95% α-1,4- and 5% α-1,6-bond content In addition, hydrogen bonding between O3 and O2 atoms of adjacent sugar residues generates a helical twist of the polymer Amylose in starch is present as double-helical A- and B-amyloses, and the single-helical V-amylose V-amylose is found naturally in non-A and non-B segments of amylose and. .. end of a starch polymer to liberate either glucose or maltose from the chain end Examples of exoamylases are β-amylases (EC 3.2.1.2), glucoamylases (EC 3.2.1.3) and α-glucosidases (EC 3.2.1.20) β -amylase cleaves α-1,4 glycosidic bonds specific to maltose, glucoamylase produces glucose with β-anomeric configuration, and α-glucosidase produces glucose with α-anomeric configurations Glucoamylase and α-... cleave both α-1,4 and α-1,6 glycosidic bonds These 20 amylolytic enzymes display multi-domain organization, and belong to either of the following GH families: α-amylases, GH13 and GH57; GH14, β-amylases; and GH15, glucoamylases The three types of amylolytic enzymes do not share any sequence similarity, and about 10% carry a non-catalytic domain that can bind to raw starch Whereas α-amylases are retaining... starch-degrading enzymes can be divided in to four groups based on the reactions they catalyze: exoamylase; (iii) debranching enzymes; and (iv) (i) endoamylase; (ii) transferases (Fig 1.8) Endoamylases, the most common member being α -amylase (EC 3.2.1.1), perform random cleavage of the α-1,4 glycosidic bonds in both amylose and amylopectin to produce shorter linear and branched oligosaccharides Exoamylases cleave... is in the range 1000 to 6000 glucose units 18 (Fig 1.6 b) Amylopectin, which refers to the branched polymer, is linear with typical length of 12-120 glucose units linked by α-1,4 bonds The branches are linked at the branch points by α-1,6 bonds, and range in size from 15 to 45 glucose units (Fig 1.6 c) The length of both α-1,4 backbone and α-1,6-linked branches varies depending on the botanical origin... structure of the halotolerant and thermostable α -amylase AmyB from Halothermothrix orenii at 2.3 Å resolution In addition, the structures of AmyB in complex with a nonasaccharide resulting from transglycosylation of the inhibitor acarbose at 1.35 Å resolution, and the 2.2 Å structure of the enzyme in complex with hydrolysis products of maltoheptaose have been determined The 1.35 Å structure is hitherto at... have an important role in the stabilization of thermophilic proteins, they are not the sole determinants of thermostability A major shortcoming of most studies that attempt to explain the mechanisms of thermal adaptation is that only small sets of proteins are compared and analyzed However, well into the post-genomic era it is now possible to take full advantage of genomic data and the outcomes of structuralgenomics... is composed of eight twisted parallel β strands arranged close together into a barrel Each parallel β strand is connected to an α helix that packs onto the β sheet on the outside of the barrel The structure can be regarded as consisting of repeated βαβ motifs TIM barrels are observed predominantly in enzymes, and a canonical feature is the location of the active sites at the C-terminal end of the barrel... retaining enzymes, β-amylases and glucoamylases are inverting α -1,6 hydrolysis isoamylase/amylopullulanase α -1,6 hydrolysis glucan branching enzyme α -1,4 hydrolysis α -1,6 transferase glucoamylase/ α-glucosidase cyclodextrin glycosyltransferase α -1,4 hydrolysis α -1,4 transferase β -amylase/ maltogenic amylase amylomaltase α -amylase Figure 1.8 Enzymes involved in starch processing Grey and yellow rings . CRYSTAL STRUCTURE OF AMYB, AN α- AMYLASE FROM HALOTHERMOTHRIX ORENII, AND COMPARISON WITH ITS HOMOLOGS Tien Chye Tan Submitted 14 April 2007 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. the revised version of the thesis, and advice on revision of the thesis. And for pulling my brain out of the gutter and getting it to stay focused. Prof Birte Svensson and Dr Karen Marie Jakobsen. by means of molecular replacement, the crystal structure of the halotolerant and thermostable α -amylase AmyB from Halothermothrix orenii at 2.3 Å resolution. In addition, the structures of