CHAPTER CRYSTAL STRUCTURE DETERMINATION OF AMYA FROM HALOTHERMOTHRIX ORENII 2.1 INTRODUCTION Some organisms can live in extreme conditions like temperatures that are close to the boiling point of water, pressures that are many hundred times that of atmospheric pressure and salinity that is orders of magnitude above typical physiological conditions. These extremophiles use several cellular and structural adaptation mechanisms to be able to survive, actively grow and propagate in extreme environments. It has been reported that macromolecules, especially proteins from extremophiles and mesophiles, have the same overall fold and molecular mechanism for their function (Vieille et al, 1995; Russell et al, 1997; Bauer et al, 1998). However, certain structural features make these proteins stable and optimally active under extreme conditions. Considerable effort has been made to date to understand the molecular mechanism of adaptation of proteins from thermophilic and halophilic organisms at the atomic level. 2.1.1 Thermophilic protein stability With the exception of phylogenetic variations, what differentiates thermophilic and mesophilic enzymes is only the temperature ranges in which they are stable and active. Otherwise, thermophilic and mesophilic enzymes are highly similar. The sequences of homologous thermophilic and mesophilic proteins are typically 40 to 85% similar (Davies et al, 1993), their three-dimensional structures are superimposable (Auerbach et al, 1998; Tahirov et al, 1998) and they have the same 26 catalytic mechanism (Bauer et al, 1998). Thermophilic proteins are the most widely studied due to their extreme stability and ease of purification. From these studies it appears that thermal stability is not determined by any single factor but the combination of several factors, each with a relatively small effect (Perutz and Raidt, 1975; Argos et al, 1979; Vogt et al, 1997; Jaenicke and Bohm, 1998; Shih and Kirsch, 1995). These factors include the following. 2.1.1.1 Amino acid composition The amino acid composition of a protein has long been thought to be correlated to its thermostability. The first statistical analysis comparing amino acid compositions in mesophilic and thermophilic proteins indicated the trends toward substitutions such as Glycine to Alanine and Lysine to Arginine. A higher Alanine content in thermophilic proteins was supposed to reflect the fact that Alanine was the best helix-forming residue (Argos et al, 1979). As more experimental data accumulate, in particular complete genome sequences, it is becoming obvious that "traffic rules of thermophilic adaptation cannot be defined in terms of significant differences in the amino acid composition" (Bohm and Jaenicke, 1994). The comparison of residue contents in hyperthermophilic and mesophilic proteins based on the genome sequences of mesophilic and hyperthermophilic organisms shows only minor trends. 2.1.1.2 Hydrophobic interactions The hydrophobic effect is considered to be a major driving force of protein folding. Hydrophobicity drives the protein to a collapsed structure from which the native structure is defined by the contribution of all types of forces (Dill, 1990). 27 Thermophilic proteins normally have extensive hydrophobic interactions and reduced water accessible hydrophobic surface area compared to their mesophilic counterparts (Wigley et al, 1987). 2.1.1.3 Disulfide bridges Disulfide bridges are believed to stabilize proteins mostly through an entropic effect by decreasing the entropy of the protein's unfolded state (Matsumura et al, 1989). The entropic effect of the disulfide bridge increases in proportion to the logarithm of the number of residues separating the two bridged cysteines. 2.1.1.4 Aromatic interactions Aromatic-aromatic interactions (aromatic pairs) are defined by a distance of less than 7.0 Å between the phenyl ring centroids. Aromatic amino acid interactions are known to be one of the determinants of thermal stability in thermophilic proteins (Kannan and Vishveshwara, 2000; Serrano et al, 1991). A pair of aromatic interactions contributes between -0.6 and -1.3 kcal/mol to protein stability (Serrano et al, 1991). 2.1.1.5 Ion-pair Salt bridges are formed by spatially proximal pairs of oppositely charged residues in native protein structures. Often salt-bridging residues are also close in the protein sequence and fall in the same secondary structural element, building block, autonomous folding unit, domain, or subunit, consistent with the hierarchical model for protein folding. Salt bridges are rarely found across protein parts which are joined by flexible hinges, a fact suggesting that salt bridges constrain flexibility and motion. 28 While conventional chemical intuition expects that salt bridges contribute favorably to protein stability, recent computational and experimental evidence shows that salt bridges can be stabilizing or destabilizing. Due to systemic protein flexibility, reflected in small-scale side-chain and backbone atom motions, salt bridges and their stabilities fluctuate in proteins. At the same time, genomewide amino acid sequence composition, structural, and thermodynamic comparisons of thermophilic and mesophilic proteins indicate that specific interactions, such as salt bridges, may contribute significantly towards the thermophilic-mesophilic protein stability differential. Ion pair networks are energetically more favorable than an equivalent number of isolated ion pairs because for each new pair the burial cost is cut in half: only one additional residue must be desolvated and immobilized (Yip et al, 1995). 2.1.1.6 Metal binding Metals have long been known to stabilize and activate enzymes. In proteins, metal ions are coordinated, usually by lone pair electron donation from oxygen or nitrogen atoms. Some thermophilic and hyperthermophilic enzymes have been reported that contain metal atoms that are not present in their mesophilic homologs. Experiments have shown that metal binding can contribute - kcal/mol to stability. 2.1.1.7 Extrinsic parameters While most pure hyperthermophilic enzymes are intrinsically very stable, some intracellular hyperthermophilic proteins get their high thermostability from intracellular environmental factors such as salts, high protein concentrations, coenzymes, substrates, activators, polyamines, or an extracellular environmental factor such as pressure. 29 2.1.2 Halophilic protein stability Halophiles (salt-lover) can be defined as microorganisms that require high salt in a concentration range of - M to grow (Richard & Zaccai, 2000). In order to overcome the extreme osmotic pressure of these hyper saline environments, halophilic bacteria and eukaryotes accumulate mostly neutral organic compatible solutes and exclude most of the inorganic salts. In contrast, halophilic archaea balance the external high salt concentration by intracellular accumulation of inorganic ions to concentrations that exceed that of the medium. Therefore, all the cellular components of the halophilic archaea must adapt to function at the extremely high intracellular salt concentration. Halophilic proteins require a minimum of M salt concentration to be optimally active and stable. At high salt concentrations, proteins are in general destabilized due to enhanced hydrophobic interactions. Halophilic proteins have, therefore, evolved specific mechanisms that allow them to be both stable and soluble in high salt concentration. The adoptive mechanism of halophilic proteins has not been studied as extensively as thermophilic proteins due to the difficulty in purifying and crystallizing them at very high ionic strengths. Halophilic enzymes are usually very unstable in low salt concentrations. Since some of the important fractionation methods in protein chemistry, such as, electrophoresis and ion exchange chromatography, cannot be applied at high salt concentrations, the available fractionation methods for halophilic bacterial proteins are rather limited. 30 In silico analyses of the genome sequence of halophilic organisms suggest that proteins from these organisms have unique amino acid compositions. They have at least twice the number of acidic residues than basic residues (Fukuchi et al, 2003; Bieger et al, 2003). Structural insights gathered from the known halophilic crystal structures suggest that the acidic surface and the associated negative electrostatic surface potential, is one of the major stabilizing forces and is a highly conserved feature (Dym et al, 1995; Bieger et al, 2003), Fig. 2.1. Figure 2.1 The electrostatic surface potential of malate dehydrogenase from Haloarcula marismortui, a typical halophilic protein (PDB id: 1D3A). The electrostatic drawings were produced using the program GRASP. Surface colors represent the potential from -10 kBT-1 (red) to +10 kBT-1 (blue). Since all soluble halophilic enzymes have a highly negative surface charge, once folded properly, their flexibility may be achieved by repulsive forces between closely placed charged residues. The instability caused by the high surface charge density should be somehow balanced. Otherwise, the polypeptide chain will unfold. It was long believed that one of the roles of high salt concentration was to shield this high surface charge. Indeed, classical electrostatic calculations using Poisson– Boltzmann equation (Elcock and McCammon, 1998) suggest that at pH 7.0 the 31 stability of halophilic proteins is decreased by 18.2 kcal/mol on lowering the salt concentration from to 0.1 M. Using thermodynamic theories to analyze various biophysical measurements (Bonnete et al, 1993) it was calculated that, in its native state at M NaCl, halophilic malate dehydrogenase (MDH) binds approximately 200 molecules of salt and almost 3000 molecules of water. These values are significantly higher than those measured for non-halophilic proteins under the same condition and also higher than the number of salt and water molecules bound in low salt solutions in which the halophilic enzyme is unfolded. These findings are the basis for the ‘halophilic stabilization model’ for solutions in NaCl, KCl and MgCl2 (Zaccai, 1989). According to this model the tertiary and quaternary structures of native halophilic proteins co-ordinate hydrated salt ions on their surface at higher local concentrations than in the surrounding solution by specific interactions with the surface carboxyl groups. Through the binding of hydrated salt ions, water molecules would be associated with the protein structure with different local salt concentrations depending on the hydrated interactions of the particular salt. When the bulk salt concentration is reduced, salt will diffuse from the ‘quasi-crystalline’ protein-associated layer into the solvent bulk, destabilizing the protein surface and causing dissociation of the enzyme into its subunits and unfolding of the polypeptide chain. According to this model, the stabilization is enthalpy driven. The entropic penalty derived from the organization of the hydrated salt is compensated by the enthalpy of the binding of the hydrated salt to the surface carboxyl groups. This explanation for the role of salt in halophilic protein stabilization is challenged by two experimental results. First, the high resolution three-dimensional structure of Haloarcula marismortui ferredoxin (HmFd) demonstrates very clearly 32 that although the protein was crystallized from 3.8 M sodium–potassium phosphate, very few counter ions were found to be bound to the protein and when bound, they interact with the main-chain carbonyl oxygen and not with side-chain carboxylates (Frolow et al, 1996) Second, sub-millimolar concentrations of NADH can effectively replace the requirement for molar quantities of salt in the stabilization of halophilic malate dehydrogenase (MDH). Therefore, neutralization of surface charge by salt may not be required for protein stability (Irimia et al, 2003). In addition, some of the thermophilic protein determinants like metal ion binding and salt bridge networks also play a role in stabilizing halophilic proteins (Dym et al, 1995; Bieger et al, 2003). 2.1.3 Poly-extremophiles Poly-extremophiles can be defined as the organisms that require more than one extreme condition for its optimal growth and survival. For example, the organism Thermosipho japonicus isolated from a deep-sea hydrothermal vent in the Okinawa area, Japan requires high temperature and high pressure for its optimal growth (Takai et al, 2000). An interesting category among this involves the organisms that require both high temperatures and high salt concentrations. Extremophilic microbes of this kind are rare in nature and those isolated so far are difficult to handle in routine laboratories. To date only two such organisms have been reported, namely, Halothermothrix orenii and Thermohalobacter barrensis (Mijts and Patel, 2002; Cayol et al, 1994), both of which are members of the low G+C DNA-containing gram-positive phylum. H. orenii is a true halophilic and thermophilic anaerobic bacterium that was isolated from the Tunisian salt lake in the Sahara desert. It requires 33 M NaCl and 60 °C temperature for optimal growth but still shows significant growth up to M NaCl (Cayol et al, 1994). Halothermothrix orenii is a member of the family Haloanaerobiaceae, order Haloanaerobiales (Cayol et al, 1994), whereas Thermohalobacter berrensis, a member of the order Clostridiales, grows readily at 70 °C in the presence of 15% NaCl (Cayol et al, 2000). These organisms drastically differ from other extremophiles as they handle both physical and chemical extremes at the same time. The study of the molecular adaptation of proteins at more than one extreme i.e. poly-extreme condition is very important in the understanding of the biology of these organisms. A few specific questions may be posed in this halo- and thermophilic group of poly-extremophilies. 1) Are the structural adaptations the same as those found in halophilic and thermophilic organisms or would it be a completely new set of adaptations? 2) In the former case, would these adaptations be a simple addition of structural features or are there complex interactions of these features? 3) What specific differences exist in the poly-extreme adaptations? 4) What is the impact of the structural adaptations on the function and mechanism of the proteins? To address these questions we have undertaken a biophysical study of AmyA, a secretory α-amylase (Mijts and Patel, 2002) from Halothermothrix orenii. AmyA is an endo-acting α-amylase and randomly cleaves the α-1,4-glycosidic linkages present in starch and its constituent polysaccharides amylose and amylopectin (Mijts and Patel, 2002). AmyA is active in a broader salt concentration ranging between and M NaCl. However, it has optimal activity at M NaCl concentration and temperature above 65 °C, similar to the optimal conditions for H. orenii growth. The obligatory 34 requirement of salt at molar levels and temperature above 60 °C makes AmyA a true halo-thermophilic enzyme. 2.1.4 The α-amylases: Alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) are Endo-acting enzymes that catalyze the hydrolysis of alpha-1,4-glycosidic bonds in starch and related poly and oligosaccharides. The α-amylase family comprises a group of enzymes with a variety of different specificities that all act on one type of substrate, being glucose residues linked through an α–1,1, 1,4, or 1,6 glycosidic bond. The members of this family share a number of common characteristics but at least 21 different enzyme specificities are found within the family. These differences in specificity are based not only on subtle differences within the active site of the enzymes but also on the differences within the overall architecture of the enzymes. The α-amylase family can roughly be divided into two subgroups: the starch-hydrolyzing enzymes and the starch-modifying or transglycosylating enzymes. The hydrolases and transferases that constitute the α-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (β/α)8-barrel with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities 35 Table 2.2 Refinement statistics of AmyA at low salt. Values in parentheses are for the last resolution shell. ------------------------------------------------------------------------------------Refinement Resolution range (Å) 10–1.6 (1.7–1.6) |F|/σ(|F|) >0 Protein atoms 4025 Water molecules 551 Calcium Chloride R work (%)1 20.1(23.6) R free (%)2 22.1(26.6) Reflections (working/test) 56,419/6,331 RMS deviations from ideal stereochemistry Bond lengths (Å) 0.006 Bond angles (º) 1.27 B factors Mean B factor (Protein) (Å ) Mean B factor (Water) (Å ) Mean B factor (Ions) (Å ) 17.9 31.5 13.7 ---------------------------------------------------------------------------------------------1 Rwork =∑hkl||Fo(hkl)| – |Fc(hkl)|| / ∑hkl|Fo(hkl)|. 2Rfree is equivalent to Rwork and is calculated for a randomly chosen 10% of reflections but omitted from the refinement process. Over expression construct was transformed into E. coli BL21 (DE3) cells and the cells were grown in LB broth supplemented with 100 μg ml-1 Ampicillin. Over expression 43 of AmyA was induced with 0.1 mM IPTG for overnight at 30 ºC. Cells were harvested by centrifugation, lyzed in 50 mM Tris (pH 8.0), mM DTT and 500 mM NaCl. The over expressed AmyA protein was purified using Sepharose 4B column chromatography following which the GST tag was cleaved with the precision protease purchased Amersham. The resulting protein preparation was purified by a heat step consisting of incubation at 65 ºC for 30 minutes and the denatured impurities resulted after the heat incubation were removed by centrifugation. The Amya protein was further purified using Superdex-75 gel filtration column chromatography and dialyzed against 50 mM Tris (pH 8.0) containing different NaCl concentration depending upon the experimental need. 2.2.5 Enzymatic assays Amylase activity assays were performed by using the EnzCheck Amylase Activity Assay kit (Molecular Probes). α-amylase from Bacillus sp, which was provided with the kit, served as a positive control enzyme. One unit is defined as the amount of enzyme required to liberate mg maltose from starch in minutes under assay conditions. The data presented are the means of four individual readings, collected from four individual experiments. 2.3 RESULTS 2.3.1 Overview of AmyA structure AmyA consists of a single polypeptide chain of 488 residues and comprises a total of 18 α-helices and 20 β-strands. The overall fold of AmyA consists of three distinct domains, A, B and C (Fig. 2.3). The central A domain forms the (β/α)8 TIM barrel structure through residues 27–131 and 198–436. The symmetry of the central 44 (β/α)8 TIM barrel is interrupted by domain B that consists of 66 residues (132–197), which is inserted between strand β5 and helix α5. Domain B consists of two α-helices, two beta strands and a highly flexible and long loop protruding close to the active site. On a structural level, domain B is the least conserved segment among α-amylases from different origins and other carbohydrate-processing enzymes. Figure 2.6 A ribbon diagram of the AmyA molecule. The three domains are represented in different colors and the calcium ions are shown as red spheres. The B domain of AmyA is very similar to that of oligo-1,6-glucosidase and isomaltulose synthase, Fig. 2.4 (Watanbe et al, 1997; Zhang et al, 2003). These two enzymes are structurally very similar to AmyA. Even though all three enzymes possess the glycosidic activity, the glucosyltransferase activity is present only in 45 Figure 2.7 Comparison of the domain B structure of AmyA (green) with that of oligo-1,6-glucosidase (PDB entry 1UOK, blue) and isomaltulose synthase (PDB entry 1M53, magenta). oligo-1,6-glucosidase and isomaltulose synthase and is absent in AmyA. A loop in domain B that contains residues 159-173, which reside in the vicinity of the catalytic site and have very high temperature factors, shows multiple occupancies in the electron density map and does not have sequence homology with α-amylases. However, this loop has high sequence homology with the carbohydrate binding loop of a neuraminidase (Burmeister et al, 1992), suggesting that this loop could be involved in substrate binding. Domain C is located exactly opposite to domain B on the other side of domain A. The C-terminal domain, which consists of 79 residues (437–515), forms a separated folding unit, exclusively made up of β-sheets. Eight of the ten strands fold 46 into a β-sandwich structure with the ‘Greek key’ topology. Domain C is also highly conserved among α-amylase family enzymes and is reported to be involved in substrate binding (Zhang et al, 2003). However, the clear mechanism is not yet known. The overall fold and domain structure of AmyA are similar to those of the members of family 13 glycoside hydrolases. Despite these similarities, a key and significant attribute that has been identified from sequence alignment and structural comparison of the AmyA structure with other homologous hydrolases is that while most of the conserved residues are buried, the surface accessible residues are unique (Fig. 2.5). There are marked differences between the loops connecting the secondary structure elements of the members of the α-amylase family of enzymes, especially those that surround the active site. These loops might be responsible for the distinct catalytic properties among these enzymes. Comparison of the loop structures between members of the family reveals that AmyA has some novel extended loops that bind calcium ions. The calcium binding loop that contain residues 66-73 and the loop containing residues 230-239 immediately after the catalytic residues Arg222 and Asp224 are absent in other α-amylases. Structural alignment results from the DALI server (Holm and Sander, 1993) show that the tertiary structure of AmyA has the highest structural similarity to oligo1,6-glucosidase from Bacillus cereus (Watanbe et al, 1997). A total of 468 residues (all atoms) could be superimposed with a root mean square deviation (RMSD) of 2.79 Å. RMS differences for the individual domains(all atoms) have been calculated by using the program TM-align (Zhang et al, 2005) and have values of 2.52, 1.78 and 2.43 Å for the A, B and C domains, respectively. 47 Figure 2.8 The sequence alignment of AmyA. The amino acid sequence alignment of AmyA with mesophilic α-amylase from Bacillus Cereus (1UOK), thermophilic αamylases from Thermus Sp (1SMA) and Thermoactinomyces vulgaris R- 47 (1JI2). Conserved residues are in blue boxes; identical residues among all the four sequences are indicated by white letters with red background and similar residues are indicated by red letters. The secondary structure elements of AmyA are shown on top of the AmyA sequence. Surface accessibility of residues is shown at the bottom of the aligned sequences. The most accessible residues are shown in dark blue and buried residues are color coded in white. 48 2.3.2 Catalytic site Sequence alignment with various α-amylases shows that the well conserved catalytic triad, Asp224, Glu260, Asp330 (AmyA numbering), is located at the center of the barrel (Fig. 2.6). The overall conservation of other catalytic residues and water molecules in conjunction with the previous structural and biochemical analyses suggests that the enzymatic mechanism of AmyA is very similar to the doubledisplacement catalytic mechanism of other known α-amylases (Strobl et al, 1998). Figure 2.9 A stereo view of the active site. A stereo view of the active site cleft at the center of the TIM barrel domain is shown. The conserved catalytic residues and water molecules are shown as sticks and spheres, respectively. 2.3.3 Calcium binding The AmyA structure binds to two calcium ions. The calcium sites have been identified from the difference Fourier map as strong peaks with appropriate coordination atoms. The ions are hexa-coordinated and the geometry is described as a distorted octahedron, which is often observed in the metal ion binding sites of metallo 49 proteins. The residues and the water molecules that make coordination bonds with the two calcium ions are listed below with measured distances, Table 2.3. Table 2.3 Coordinating atoms of calcium and distances. Calcium No Interacting Atoms ASP 44 OD1 ASP 46 OD1 ASP 48 OD1 ILE 50 O ASP 52 OD2 WAT 27 Distance in Å 2.47 2.49 2.50 2.45 2.48 2.70 Calcium No Interacting Atoms ASP 65 OD1 ASP 67 O THR 70 O THR 70 OG1 ASP 73 OD1 WAT 69 Distance in Å 2.59 2.50 2.64 2.58 2.59 2.70 Ca 1, the calcium ion, which is coordinated by residues within 44 to 52, is present in most α-amylases, Figs. 2.7, 2.8. Also, a novel calcium site, Ca2, is also present in AmyA. This calcium ion is not present in other amylase family enzymes. In the previous studies it has been shown that heating will release calcium from the protein. Even though no calcium salts were added during enzyme purification or crystallization and AmyA was heated up to 65 °C for 30 minutes during purification, the presence of calcium ions in the structure indicates that these calcium binding pockets have very high affinity for calcium. However, the conserved calcium binding 50 sites present in other α-amylases at the interface between domains A and B are missing in AmyA. Figure 2.10 A stereo view of calcium binding. A stereo representation of a calcium binding loop is shown with the electron density. The 2Fo–Fc electron density omit map is drawn at the 1.3 σ contour level. Side chains of the amino acids that make coordination bonds with the calcium are shown as sticks and the calcium and water molecule are shown as pink and red spheres, respectively. 51 Figure 2.11 Calcium binding loop. A stick and ribbon model representation of a calcium binding loop. Side chains of the amino acids that make coordination bonds with the calcium are shown as sticks and the calcium and water molecule are shown as pink and red spheres, respectively. 2.3.4 AmyA does not require acidic surface Solvent exposed regions of proteins play a major role for protein stability at extreme environments. All known halophilic protein structures possess an overall acidic surface. This conserved acidic surface of halophilic proteins enhances the stability of the protein by increasing solvation through an increased water binding capacity (Fukuchi et al, 2003; Bieger et al, 2003). Surprisingly, we notice that unlike all the other halophilic protein structures solved to date (Fukuchi et al, 2003; Dym et al, 1995; Bieger et al, 2003), AmyA lacks the excess of acidic residues on the surface 52 and both positively and negatively charged residues are distributed equally (Table 2.4). The density of acidic residues on the surface of AmyA is one per 419 Å2. In comparison, other halophilic proteins have much higher surface density of acidic residues at one per 128-246 Å2 and non-halophilic proteins have one per 350-400 Å2 (Bieger et al, 2003). AmyA contains even smaller number of acidic residues on its surface when compared to its mesophilic and thermophilic counterparts (Table 2.4) and also the density at which these residues are present is also reduced when compared to its close homologues (Table 2.5). Despite the lack of an acidic surface, AmyA retains 90% of its optimum activity and is stable at M NaCl concentration (Mijts and Patel, 2002). Genomewide random sequence analysis of H. orenii has identified a surprising lack of excess acidic amino acid residues in most of its proteins (Mijts and Patel, 2001). Calculation of electrostatic surface potential reflects that AmyA lacks the highly electronegative surface potential that is present in halophilic proteins (Fig. 2.9). The cavity formed in the (β/α)8-barrel domain is the most electronegative feature of the surface. This is mainly due to the presence of the amylase specific negatively charged residues Asp128, Asp224, Glu260, and Asp330 that are known to be involved in substrate binding and catalysis (Abad et al, 2002). 53 Table 2.4 Exposure of charged residues on various amylase surfaces Organism No. of Residue s Halothermothrix 515 Thermus Sp pI Asp/Glu Surface charge Arg/Lys His Out In Out In Out In Out In 5.34 +0.5 -8.5 48 23 45 11 588 5.52 -7.5 -0.5 66 19 51 15 15 Thermoactinomyces Vulgaris R- 47 585 5.52 +5.5 -11.5 61 24 60 11 13 Bacillus Cereus 558 4.63 -23.5 -8.5 72 29 44 17 Orenii Sequence statistics for the protein surface of amylases. The assignment whether a residue is exposed to the outer surface (out) or the inner surface (in) was based on the crystal structure and calculated by the WHAT IF program suit (Vriend, 1990). The estimation of overall surface charges was calculated by assuming fully ionized states for Asp, Glu, Lys and Arg, and +0.5 charges per His 54 Table 2.5 Statistics of surface densities per acidic amino acid residue of various amylases No. of residu es Total surface area (Å2) Surface densities per acidic amino acid residue (Å2) Halothermothrix Orenii 515 20150.11 419.8 Thermus Sp 588 23681.31 358.8 Thermoactinomyces Vulgaris R- 47 585 23849.23 390.97 Bacillus Cereus 558 21783.51 302.55 Organism 55 Figure 2.12 The surface property of AmyA (a) The molecular surface of AmyA. Positive and negative groups are drawn in blue and red, respectively. (b) The electrostatic surface potential of AmyA. The electrostatic drawings were produced using the program GRASP (Nicholls et al, 1991). Surface colors represent the potential from -10 kBT-1 (red) to +10 kBT-1 (blue). 2.3.5 Enzyme activity The N-terminal signal peptide cleaved AmyA was purified by gel filtration and tested for activity. It shows a specific activity of 620 units mg-1, which is much higher compared to the previously reported AmyA activity of 23 units mg-1 (Mijts and 56 Patel, 2002). The optimal activity was observed at M NaCl and at 65 °C, which is close to the optimal growth condition for H. orenii. In the previous report, partially purified AmyA that contained the signal peptide with a His-tag was used for the activity assay. From the crystal structure of AmyA it appears that the His-tag and Nterminal signal peptide are likely to reside in the vicinity of the active site and may account for the difference in the reported specific activities. However, consistent with the previous report, AmyA shows activity in the entire salinity range (Fig. 2.10). The activity profiles of AmyA at different temperatures and pH are shown in Figs. 2.11 and 2.12. Figure 2.13 AmyA activity profile. Activity assay of AmyA using 20 mM Tris (pH 7.5), 1mM CaCl2 and different NaCl concentrations at 65 °C. 57 AmyA activity at different Temperature 700 Units/mg 600 500 400 300 200 100 0 25 50 75 100 Temperature in Celcius Figure 2.14 Activity profile of AmyA at different temperature. Activity assay of AmyA using 20 mM Tris (pH 7.5), 1mM CaCl2 and M NaCl concentrations at different temperature. AmyA activity at different pH 700 Units/mg 600 500 400 300 pH Figure 2.15 Activity profile of AmyA at different pH. Activity assay of AmyA using 20 mM Tris (pH 7.5), 1mM CaCl2 and M NaCl concentrations at different pH. 58 [...]... calcium ions are listed below with measured distances, Table 2. 3 Table 2. 3 Coordinating atoms of calcium and distances Calcium 1 No Interacting Atoms 1 2 3 4 5 6 ASP 44 OD1 ASP 46 OD1 ASP 48 OD1 ILE 50 O ASP 52 OD2 WAT 27 Distance in Å 2. 47 2. 49 2. 50 2. 45 2. 48 2. 70 Calcium 2 No Interacting Atoms 1 2 3 4 5 6 ASP 65 OD1 ASP 67 O THR 70 O THR 70 OG1 ASP 73 OD1 WAT 69 Distance in Å 2. 59 2. 50 2 .64 2. 58 2. 59... Celcius Figure 2 .14 Activity profile of AmyA at different temperature Activity assay of AmyA using 20 mM Tris (pH 7.5), 1mM CaCl2 and 2 M NaCl concentrations at different temperature AmyA activity at different pH 700 Units/mg 60 0 500 400 300 4 5 6 7 8 9 pH Figure 2 .15 Activity profile of AmyA at different pH Activity assay of AmyA using 20 mM Tris (pH 7.5), 1mM CaCl2 and 2 M NaCl concentrations at different... report, AmyA shows activity in the entire salinity range (Fig 2 .10 ) The activity profiles of AmyA at different temperatures and pH are shown in Figs 2 .11 and 2 . 12 Figure 2 .13 AmyA activity profile Activity assay of AmyA using 20 mM Tris (pH 7.5), 1mM CaCl2 and different NaCl concentrations at 65 °C 57 AmyA activity at different Temperature 700 Units/mg 60 0 500 400 300 20 0 10 0 0 0 25 50 75 10 0 Temperature... contains one molecule per asymmetric unit All data were indexed, integrated and scaled using the programs DENZO and SCALEPACK (Otwinowski and Minor, 19 97) The data collection and crystallographic statistics are summarized in Table 2 .1 40 Figure 2. 5 lAmyA crystal picture Crystals of AmyA, with maximum dimensions of 0 .1 x 0 .1 x 0 .6 mm Table 2 .1 Crystal parameters and data collection statistics of AmyA at. .. after the catalytic residues Arg 222 and Asp 224 are absent in other α-amylases Structural alignment results from the DALI server (Holm and Sander, 19 93) show that the tertiary structure of AmyA has the highest structural similarity to oligo1 ,6- glucosidase from Bacillus cereus (Watanbe et al, 19 97) A total of 468 residues (all atoms) could be superimposed with a root mean square deviation (RMSD) of 2. 79 Å... Refinement Resolution range (Å) 10 1. 6 (1. 7 1. 6) |F|/σ(|F|) >0 Protein atoms 4 025 Water molecules 5 51 Calcium 2 Chloride 0 R work (% )1 20 .1( 23 .6) R free (% )2 22 .1( 26 . 6) Reflections (working/test) 56, 419 /6, 3 31 RMS deviations from ideal stereochemistry Bond lengths (Å) 0.0 06 Bond angles (º) 1. 27 B factors 2 Mean B factor (Protein) (Å ) 2 Mean B factor (Water) (Å ) 2 Mean B factor (Ions) (Å ) 17 .9 31. 5 13 .7... stable and commercially useful proteins In this thesis, we report the structure of AmyA at both low and high salt concentrations at 1. 6 and 1. 83 Å resolution, respectively The analysis of AmyA structure reveals a novel surface feature and its implications for stability under poly- extreme conditions We also report the biophysical characterization studies of AmyA under a 38 broad salinity range and this... estimation of overall surface charges was calculated by assuming fully ionized states for Asp, Glu, Lys and Arg, and +0.5 charges per His 54 Table 2. 5 Statistics of surface densities per acidic amino acid residue of various amylases No of residu es Total surface area ( 2) Surface densities per acidic amino acid residue ( 2) Halothermothrix Orenii 515 2 015 0 .11 419 .8 Thermus Sp 588 2 36 81. 31 358.8 Thermoactinomyces... were harvested by centrifugation at 5000g for 10 minutes and resuspended in 20 ml phosphate buffer [20 mM sodium phosphate (pH 7.8), 500 mM NaCl] The cells were lysed at 4 °C using a French press at 6. 9 MPa DNaseI was added to the lysate at a concentration of 5 µg ml -1 and the sample was incubated on ice for 30 minutes to increase the precipitation of heat denatured protein Any insoluble material was... The domain architecture of α-amylases 2 .1. 4 .2 The catalytic mechanism of amylases: Catalytic steps in glycoside bond cleavage in retaining enzymes The proton donor protonates the glycosidic oxygen and the catalytic nucleophile attacks at C1 leading to formation of the first transition state The catalytic base promotes the attack of the incoming molecule ROH (water in hydrolysis or another sugar molecule . picture. Crystals of AmyA, with maximum dimensions of 0 .1 x 0 .1 x 0 .6 mm. Table 2 .1 Crystal parameters and data collection statistics of AmyA at low salt. Values in parentheses are for the last resolution. (Bonnete et al, 19 93) it was calculated that, in its native state at 4 M NaCl, halophilic malate dehydrogenase (MDH) binds approximately 20 0 molecules of salt and almost 3000 molecules of water (Kannan and Vishveshwara, 20 00; Serrano et al, 19 91) . A pair of aromatic interactions contributes between -0 .6 and -1. 3 kcal/mol to protein stability (Serrano et al, 19 91) . 2 .1. 1.5 Ion-pair