MINIREVIEW Implication for buried polar contacts and ion pairs in hyperthermostable enzymes Ikuo Matsui and Kazuaki Harata Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan Introduction Hyperthermophiles grow optimally at temperatures of 80–110 °C [1]. Only represented by bacterial and archaeal species, these organisms have been isolated from all types of terristerial and marine hot environ- ments. Some of the enzymes from hyperthermophiles are active at temperatures as high as 110 °C and above [2]. Recently, much effort has been directed towards the isolation and characterization of enzymes from hyperthermophilic archaea. Interest in these enzymes has increased because of their potential biotechnolo- gical applications [3,4] and because of the need for a better understanding of their intrinstic heating and denaturing resistance. Elucidating the stabilizing mechanisms has been one of the greatest challenges in biochemistry and biotechnology [3–5]. This minireview encompasses the molecular determi- nants of protein stability, and compares the various molecular structures of amino acid aminotransferase (AT), b-glycosidases (BG), and a-amylases (AM), from different origins, including hyperthermophiles, thermo- philes living at around 65–75 °C, and mesophiles living at room temperature. The three representative enzymes, AT, BG, and AM were selected due to the numerous structural information and stability data Keywords accessible surface area; buried polar contact; hyperthermophilic archaea; hyperthermostable enzyme; ion pair; molecular structure; Pyrococcus; subunit interaction; thermostability Correspondence I. Matsui, Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305, Japan Fax: +81 29 8616151 Tel: +81 29 8616142 E-mail: ik-matsui@aist.go.jp (Received 28 February 2007, accepted 17 May 2007) doi:10.1111/j.1742-4658.2007.05956.x Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology. However, the structural basis of thermostability is still not fully characterized. Ionizable residues play essen- tial roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. The anal- ysis revealed that the remarkably increased number of internal polar con- tacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120 °C for hyperthermophilic enzymes from the genus Pyrococcus. These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs. Abbreviations AM, a-amylase; AT, aminotransferase; BG, b-glycosidase; DERA, 2-deoxy- D-ribose-5-phosphate aldolase; DSC, differential scanning calorimetry; GluDH, glutamate dehydrogenase; T m , melting temperature. 4012 FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS available from widely distributing origins, including hyperthermophiles, thermophiles and mesophiles. Background: there is no single dominating factor for the thermostability of proteins Many crystal structures of hyperthermophilic enzymes have been reported, and several factors responsible for their extreme thermostability have been suggested. It was proposed that the stability of a protein could be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding [6]. Proline reduces the flexibility of the polypeptide chain. The mutations, Gly fi Xaa or Xaa fi Pro should decrease the entropy of a protein’s unfolded state and stabilize the protein. A number of the thermophilic and hyperthermophilic proteins also use this stabiliza- tion mechanism [1]. The stabilizing role of a large and more hydrophobic core was proposed based on experi- mental evidence obtained using chimeric methanocco- cal adenylate kinases [7]. The stability properties of the chimera constructed from the Methanococcus voltae and Methanococcus jannaschii adenylate kinases indi- cated that cooperative interaction within the hydro- phobic protein core plays an integral role in increasing the thermalstability. Another potential stabilizing fac- tor is the high packing density of the molecule. A com- parison of the hyperthermophilic aldehyde ferredoxin oxidoreductase with the mesophilic aldehyde ferre- doxin oxidoreductase revealed that the former has a small solvent-exposed surface area [8]. A comparison of citrate synthases from a hyperthermophile (Pyrococcus furiosus), thermophile (Thermoplasma acidophilum) and mesophilic organism (pig) indicated that increased compactness of the enzyme might be one of the major factors required for its thermostability [9]. An increase in the number of ion pairs and hydro- gen bonds is also important for thermostability. The crystal structure of glyceroaldehyde-3-phosphate dehy- drogenase from a thermophile, Thermus aquaticus, has been compared with that of three glyceroaldehyde- 3-phosphate dehydrogenases of different origins [10]. From the results, a strong correlation between thermo- stability and the number of hydrogen bonds between charged side chains and neutral partners was found. There are two reasons why proteins may use charged- neutral hydrogen bonds rather than salt links or neu- tral-neutral hydrogen bonds to stabilize protein [10]. First, the desolvation penalty associated with burying charged-neutral hydrogen bonds would be less than that of burying ion pairs because of only one charged residue being involved. Second, the enthalpic reward of charged-neutral hydrogen bonds is greater than that of neutral-neutral hydrogen bonds because of the charge–dipole interaction. By a structure comparison of [Fe 3 S 4 ]-feredoxin from the hyperthermophilic archa- eon P. furiosus with those from the thermophile and mesophile, further significant roles of the hydrogen bonds on the hyperthermostablity have been reported [11–13]. The P. furiosus feredoxin structure shows a higher degree of hydrogen-bond network than other homologus ferredoxins, and this is believed to be the main reason for the observed increased thermostability with a denaturation temperature of over 100 °C. An increase in the number of ion pairs, especially in networks, is observed nearly in every hyperthermo- stable protein [1,14]. The structral comparison of an O 6 -methylguanine-DNA methyltransferase from a hyperthermophilic archaeon, Thermococcus kodakara- ensis, with the mesophilic counterpart from Escherichi- a coli suggested that four additional buried ion pairs between a-helices might play a key role in its stabiliz- ing mechanism [15]. The amino acid residues forming interhelix ion pairs are buried relatively more often than those of intrahelix ion pairs because the aver- aged solvent-accesible surface areas of amino acid res- idues forming inter- and intrahelix ion pairs are 39.5 A ˚ 2 and 98.9 A ˚ 2 per residue, respectively. This suggests the internal location of interhelix ion pairs in the molecule. The interhelix ion pairs in the interior of the protein presumably enhance the stability of the internal packing (tertiary structure). Furthermore, a stabilizing function has also been proposed for buried ion pairs in Thermosphaera aggregans BG (BGTa) [16]. In four different sequences of hyperthermophilic BGs (BGTa from T. aggregans,BGSs from Sulfolobus solfataricus,BGPf and b-mannosidase from P. furio- sus), 28% of the residues are strictly conserved. In the aligned sequences, a strict conservation is observed among the residues participating in forming internal ion pairs; however, only 26% of the surface ion pairs are conserved, consistent with the average sequence conservation among these sequences. The homohexa- meric structure of hyperthermophilic glutamate dehy- drogenase (GluDHPf)(t 1 ⁄ 2 , 12 h; 100 °C) from P. furiosus was compared with the mesophilic GluDHCs (t 1 ⁄ 2 , 30 min; 52 °C) from Clostridium sym- biosum. The comparison revealed that the hyperther- mostable enzyme contains a striking series of ion pair networks on the surface of the protein subunits, and partially buried at intersubunit and inter-domain interfaces, not found in the mesophilic counterparts [1,17–19]. The importance of intersubunit ion pairs to the structural stability of GluDHTk from a hyperther- mophile, Thermococcus kodakaraensis, was examined I. Matsui and K. Harata Strucural elememts providing hyperthermostability FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS 4013 by site-directed mutagenesis, involving systematic addition or removal of ion pairs [20]. These results proved the important role for the intersubunit ion pairs in stabilizing the GluDHTk molecule. However, completely different results were reported from a structural analysis of GluDHPi from a hyperthermo- phile, Pyrobaculum islandicum [21]. The number of in- tersubunit ion pairs in the homohexameric GluDHPi molecule was much smaller than that in GluDHPf or the mesophilic GluDHCs. These findings suggest that the major molecular strategy for thermostability may differ for each hyperthermophilic enzyme [21]. The significant role of the entropic effect, due to shorter surface loops, on the thermostability of tryptophan synthase a-subunit from P. furiosus (a-subunit-Pf) was reported [22]. The thermostability of the a-subunit-Pf molecule was examined by differential scanning calo- rimetry (DSC) and by comparing the molecular struc- ture with that of a mesophilic a-subunit-St molecule from Salmonella typhimurium. The DSC data indicated that the greater stability of the a-subunit-Pf molecule was not caused by an enthalpic factor. From these results, it was concluded that hydrophobic interactions in the protein interior do not contribute to the higher stability of the a-subunit-Pf molecule. The increased number of ion pairs, smaller cavity volume, and entro- pic effects due to a shorter polypeptide chains, are important in the hyperthermostability of the a-subunit- Pf molecule. A comparative analysis of the proteins Thermococ- cus kodakaraensis KOD ribulose-bisphosphate carboxy- lase ⁄ oxygenase [23], Thermotoga maritima dihydrofolate reductase [24] and phosphoribosylanthranilate isomer- ase [25], and Aeropyrum pernix 2-deoxy-d-ribose- 5-phosphate aldolase (DERA) [26] suggested that oligomerization of subunits appears to be the factor responsible for the hyperthermostability. The area of the subunit–subunit interface in the dimer of the A. pernix DERA is much larger compared with that of the E. coli enzyme. Furthermore, the A. pernix DERA has an additional N-terminal helix that induces the formation of a characteristic dimer–dimer interface. These results suggest that the hyperthermostability of the A. pernix DERA could be enhanced by the forma- tion of a unique tetrameric structure unlike the dimeric structure of the mesophilic counterparts (i.e. the E. coli enzyme) [26]. Hence, protein stability appears to be attributable to a combination of factors, which are related to each other and their contribution to vary depending on the proteins. It is proposed that there is no single dominating factor for the thermostability of proteins [14]. Evaluating buried polar contacts and ion pairs as structural elements related to the thermal stability Because intermolecular and intramolecular polar inter- actions such as hydrogen bonds [11–13] and salt link- ages [1,14–22], appeared to be major factors that are responsible for hyperthermostablility, interatomic con- tacts involving main chain peptide groups and polar side chain groups of Asp, Glu, Arg, Lys, Asn, Gln, His, Thr and Ser were counted and classified on their location inside or outside of the molecule. The number of these contacts was divided by the total number of polar contacts and the results was used to evaluate the rigidity of the core region and the hydrophilic property of the molecular surface. For oligomeric proteins, in- termolecular polar contacts between subunits were also calculated. The solvent-accessible surface area was cal- culated by the Lee and Richards algorithm (probe radius 1.6 A ˚ ) [27]. Next, the accessible surface area of a protein molecule was divided by the number of amino acid residues and used as an indicator to com- pare the compactness of the protein structure. Structural elements responsible for thermostability: increase in molecular compactness, hydrophilicity of the molecular surface, and buried polar contacts ATs have been widely applied to the large-scale bio- synthesis of unnatural amino acids, which are in increasing demand by the pharmaceutical industry for peptidomimic and other single-enantiomer drugs [28]. An aspartate aminotransferase gene homolog (ORF: PH1371) was identified by sequencing the genome of a hyperthermophilic archaeon, Pyrococcus horikoshii [29,30]. The gene (ArATPh) was expressed in E. coli, and the product was purified to homogeneity. The enzyme ArATPh was proven to be an aromatic amino- transferase [31]. ArATPh is one of the most thermosta- ble aminotransferases reported to date far, with a melting temperature (T m ) of 120 °C. The crystal struc- ture of ArATPh was determined at a resolution of 2.1 A ˚ [31] and shown in Fig. 1 as protein databank accession code (PDB ID): 1dju. ArATPh has a homo- dimeric structure in which each subunit has two domains similar to other aminotransferases. As shown in Fig. 1, the ArATPh structure is more compact due to shortened loops (colored in green) compared to those observed in thermophilic (Thermus thermophilus, PDB ID: 1bjw) and mesophilic (E. coli, PDB ID: 1ars). According to the thermodynamic database of proteins Strucural elememts providing hyperthermostability I. Matsui and K. Harata 4014 FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS and mutants (ProTherm; http://gibk26.bse.kyutech. ac.jp/jouhou/protherm/protherm.html) [32], the highest T m of an enzyme measured directly by DSC was 121.6 °C for cytochrome c3 from Desulfovibrio vulgaris [33], although the T m value of PhCutA1 from P. hori- koshii was reported recently to be 150 °C [34]. From the ArATPh structure, the accumulated acces- sible surface area and intermolecular polar contacts at distances shorter than 3.3 A ˚ were calculated (Table 1). Inside–inside contacts refer to amino acid residues that are buried inside the molecule and they are not sol- vent accessible, whereas surface–surface contacts refer to residues that are exposed to the solvent, even par- tially. Such parameters that measure structure features related to thermal stability were calculated for eight aminotransferase molecules derived from hyperthermo- philes, thermophiles and mesophiles, including mam- mals such as pig and human [31,35–41], and are summarized in Table 2. The optimal growing tempera- ture of each living organism, the enzyme name, the PDB ID, and the T m measured by DSC are also shown in Table 2. The accessible surface area divided by the total residue number of the dimer was used as a reference to evaluate molecular compactness. The occupancy of charged residues in the solvent-accessible area was useful to evaluate the hydrophilicity of the molecular surface. All aminotransferases listed in Table 2 are homodimers. Their Z score and rmsd values range between 14.8 and 7.0 A ˚ and between 1.07 and 2.38 A ˚ , Fig. 1. The crystal structures of ATs and BGs. The figures were produced using the program TURBO-FRODO. (Left) C a -tracing of the hyperthermophilic ArATPh dimer (PDB ID: 1dju), thermophilic AT dimer (from Thermus thermophilus, PDB ID: 1bjw), and mesophilic AT dimer (from Escherichia coli, PDB ID: 1ars). a-Helices, b-sheets and loops are colored in pink, blue and green, respec- tively. The cofactors, pyridoxal 5¢-phosphate (PLP) molecules covalently binding to the essential Lys residue, are shown with a space filling model. (Right) C a -tracing of the hyperthermophilic BGPh molecule (PDB ID: 1vff) and mesophilic BG (from Paenibacillus polymyxa, PDB ID: 1bga). The model is viewed along the axis of the barrel. a-Helices, b-sheets and loops are colored in pink, blue and green, respectively. Table 1. The solvent-accessible surface area and intramolecular polar contacts less than 3.3 A ˚ of an aromatic amino acid amino- transferase (ArATPh) as a dimer from an hyperthermophilic archa- eon, Pyrococcus horikoshii. Total residues a Total accessible surface area (A ˚ 2 ) b Total buried residues 748 (100%) 20924.37 304 (40.6%) Total The number and ratio of intramolecular polar contacts less than 3.3 A ˚ Inside– inside Inside– surface Surface– surface Subunit– subunit 1030 (100%) 188 (18.3%) 471 (45.7%) 371 (36.0%) 46 (4.5%) a Total residue number consisting of dimer excluding the disordered regions of the molecule. b The 86.4% of the total surface area is made up of side chains, which corresponds to 18088.29 A ˚ 2 . I. Matsui and K. Harata Strucural elememts providing hyperthermostability FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS 4015 Table 2. Comparison of structural similarity, melting temperature, molecular compactness, hydrophilicity of the molecular surface and intermolecular polar contact as the rigidity of the core region for various ATs of different origins, including hyperthermophiles, thermophile and mesophilic organisms. ND, not determined. Biological diversity Optimally growing temperature (°C) Enzyme name PDB ID Structure similarity a Melting temperature by DSC (°C) Accessible surface area ⁄ residue number (A ˚ 2 ) Occupancy of charged residues in the accesible surface area (%) Intermolecular polar contact less than 3.3 A ˚ Reference Homology (%) Z score rmsd (A ˚ ) Inside– inside (%) Surface– surface (%) Subunit– subunit (%) Nonsurface ionic (%) Hyperthermophile Pyrococcus horikoshii 98 Aromatic amino acid aminotransferase 1dju 100 20.5 0 120 28.0 73.3 18.3 36.0 4.5 4.3 [1,31] Pyrococcus horikoshii Human kynurenine aminitransferase II homolog 1x0m 27 10.3 1.93 ND 33.0 65.4 13.8 49.0 3.6 2.5 [35] Thermophile 70–75 Thermus thermophilus Aspartate aminotransferase 1bjw 43 14.8 1.07 ND 32.5 56.0 10.2 57.7 5.2 0.7 [1,36] Mesophilic organisms Room Escherichia coli temperature Aspartate aminotransferase 1ars 19 7.8 2.11 63 33.7 45.8 11.1 52.7 4.0 1.8 [37,42] Paracoccus denitrificans Aromatic amino acid aminotransferase 1ay4 17 7.0 2.38 ND 33.6 48.1 10.3 51.3 3.2 1.9 [38] Trypanosoma cruzi Tyrosine aminotransferase 1bw0 25 12.1 1.63 ND 31.6 52.4 9.0 48.0 3.3 2.1 [39] Pig Cytosolic aspartate aminotransferase 1ajs 18 7.1 2.29 ND 32.8 43.9 11.8 56.8 3.8 0.0 [40] Human Kynurenine aminotransferase 1w7n 30 11.4 1.77 ND 32.8 45.6 11.7 46.3 4.3 0.6 [41] a The homology (%), Z score and rmsd values against the ArATPh molecule were retrieved by protein structure matching in a macromolecular structure database (EMBL-EBI) (http:// www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver). Strucural elememts providing hyperthermostability I. Matsui and K. Harata 4016 FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS respectively, reflecting a high structural similarity to ArATPh, although the sequence similarity varied between 43% and 17%. The T m of the mesophilic ATEc from E. coli measured directly by DSC is approximately 63 °C [42], whereas that of the hyper- thermophilic ArATPh is 120 °C [31]. A comparison of the accessible surface area per amino acid residue for the enzymes (Table 2) shows that the value of ArATPh is the lowest (28.0 A ˚ 2 ), suggesting tightest molecular packing. Another prominent feature of ArATPh is the largest occupancy of charged residues (Asp, Glu, Lys, and Arg) at its surface (up to 73.3%), indicating a hydrophilic molecular surface. Moreover, the fre- quency of buried polar contacts among all polar con- tacts in distances less than 3.3 A ˚ in the ArATPh molecule is highest (18.3%) as shown in Tables 1 and 2. By contrast, the frequency of polar contact on the surface of ArATPh is the lowest (36.0%) relative to that of the other enzymes listed in Tables 1 and 2. Interestingly, the presence of polar contact at the inter- face between the monomers is essentially the same for all enzymes tested. With an increase in the optimum growing temperature of each organism, the molecular compactness, surface hydrophilicity and ratio of buried polar contacts of each aminotransferase appears to be increased. BGs are a group of enzymes that hydrolyze the b-glycosidic linkage between carbohydrates or between a carbohydrate and a noncarbohydrate moiety. The BGs from the hyperthermophilic archaeon P. horiko- shii (BGPh) were crystallized in the presence of a neu- tral surfactant, and the crystal structure was solved at 2.5 A ˚ resolution [43] (Fig. 1). Notably, the major dif- ference of the amino acid sequence among BGPh, BGTa from T. aggregans [16], and BGSs from S. solfataricus [44], is the deletion of more than 50 residues from the BGPh sequence that are assigned to loops. As shown in Fig. 1, the overall structure of the hyperthermophilic BGPh (PDB ID: 1vff) looks very similar to that of mesophilic BGs from Paenibacillus polymyxa (BGPp) (PDB ID: 1bga). BGPh is a mem- brane-bound enzyme that is extremely thermostable and it has been shown to have high affinity for alkyl-b-glycosides. Hence, this enzyme may be used in industrial applications for the degradation of sugar derivatives and in the synthesis of various alkyl-glyco- sides via transglycosidation or ‘reverse hydrolysis’ [45]. Parameters relevant to thermal stability were calcu- lated for six BG molecules derived from hyperthermo- philes, mesophiles and plants, and are summarized in Table 3 [43–49]. The oligomeric state of the BG mole- cules listed in Table 3 varies from monomer (BGPh)to homooctamer (BGPp); however, their Z score and rmsd values range between 15.3 and 12.4 A ˚ and between 1.47 and 1.66 A ˚ , respectively. The sequence similarity varied between 35% and 30%. These data show that there is a high structural similarity between BG molecules; the main difference among them being their oligomeric state. BGPh is more thermostable than the mesophilic BGPp molecule; t 1 ⁄ 2 of BGPh at 90 °C is 15 h [45], whereas t 1 ⁄ 2 of BGPp at 35 °C has been reported to be 15 min [46]. The prevalence of charged residues at the surface of hyperthermophilic BG is higher than that of the homologus proteins from mesophilic organisms (BGPh; 56.2%, BGTa; 52.1%); this suggests that the hyperthermophilic enzyme has a more hydrophilic surface. The frequency of buried polar contacts in the BGPh molecule is the highest (14.4%) as shown in Table 3, whereas the accessible surface area per amino acid residue showed no promi- nent difference between hyperthermophilic and meso- philic BGs. The molecular compactness of BGs was calculated for the monomeric form regardless of the oligomeric state, which varied from monomer to homooctamer: with increasing thermostability, the sur- face hydrophilicity and the percentage of buried polar contacts of the BG enzymes was also increased. AMs catalyze the hydrolysis of a-(1,4)glycosidic linkages of starch components, glycogen and various oligosaccharides, and is an important industrial enzyme. The a-amylase (AMPw) from the hyper- thermophilic archaeon Pyrococcus woesei, which grows optimally at 100–103 °C, was crystallized, and the molecular structure was solved at 2.0 A ˚ resolution [50]. Many a-amylases have been isolated and charac- terized from hyperthermophiles to mesophilic organ- isms [50–58]. The AMs listed in Table 4 are monomers with known crystal structures. Except for a distant relative of a-amylase, the aminomaltase from T. aquaticus, the Z score and rmsd values of the other AMs range between 13.3 and 10.0 A ˚ and between 1.41 and 2.52 A ˚ , respectively, representing fairly good structural similarity to AMPw. The sequence similar- ity of these AMs varied between 33% and 18% (aminomaltase was excluded from the comparison). The T m value of AMPw measured by DSC is 112 °C [32], whereas the T m values of mesophilic a-amylases from a fungi, Aspergillus oryzae, and from human were reported to be 62 °C and 67.4 °C, respectively [32]. A significant difference was observed in the fre- quency of internal polar contacts of the AM mole- cules; the value of the AMPw molecule is the highest (17.1%) among the AMs listed in Table 4. Furthermore, with an increase in thermostability, the ratio of buried polar contacts also increased. How- ever, the molecular compactness, surface hydrophilicity I. Matsui and K. Harata Strucural elememts providing hyperthermostability FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS 4017 Table 3. Comparison of structural similarity, melting temperature, molecular compactness, hydrophilicity of the molecular surface, and intermolecular polar contact as the rigidity of the core region for various BGs of different origins including hyperthermophiles and mesophilic organisms. ND, not determined. Biological diversity Optimally growing temperature (°C) Enzyme name (oligomeric state) PDB ID Structure similarity a Half-life t 1 ⁄ 2 (°C) Accessible surface area ⁄ residue number (A ˚ 2 ) Occupancy of charged residues in the accesible surface area (%) Intermolecular polar contact less than 3.3 A ˚ Reference Homology (%) Z score rmsd (A ˚ ) Inside– inside (%) Surface– surface (%) Nonsurface ionic (%) Hyperthermophile Pyrococcus horikoshii 98 b-Glycosidase (monomer) 1vff 100 23.5 0 15 h (90 °C) 36.0 56.2 14.4 52.9 1.0 [1,43,45] Sulfolbus solfataricus 87 b-Galactosidase (homotetramer) 1gow 34 12.7 1.66 ND 34.6 48.3 12.1 55.4 1.3 [1,44] Thermosphera aggregans 85 b-Glycosidase (homotetramer) 1gvb 35 12.4 1.55 ND 35.6 52.1 12.7 47.2 1.5 [1,16] Mesophilic organisms Room Paenibacillus polymixa temperature b-Glucosidase (homooctamer) 1bga 34 15.3 1.49 15 min (35 °C) 33.4 43.2 11.8 49.4 1.8 [46,47] Plant (Sinapis alba) Myrosinase (homodimer) 1dwa 31 13.2 1.54 ND 33.0 48.8 11.9 51.7 1.7 [48] Plant (Trifolium repens) Cyanigenic b-Glucosidase (homodimer) 1cbg 30 13.6 1.65 ND 31.6 48.3 12.9 52.7 0.9 [49] a The homology (%), Z score and rmsd values against the BGPh molecule were retrieved by protein structure matching in a macromolecular structure database (EMBL-EBI) (http://www. ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver). Strucural elememts providing hyperthermostability I. Matsui and K. Harata 4018 FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS Table 4. Comparison of structural similarity, melting temperature, molecular compactness, hydrophilicity of the molecular surface, and intermolecular polar contact as the rigidity of the core region for various AMs of different origins including hyperthermophile, thermophiles, and mesophilic organisms. ND, not determined. Biological diversity Optimally growing temperature (°C) Enzyme name PDB ID Structure similarity a Melting temperature by DSC (°C) Accessible surface area ⁄ residue number (A ˚ 2 ) Occupancy of charged residues in the accesible surface area (%) Intermolecular polar contact less than 3.3 A ˚ Reference Homology (%) Z score rmsd (A ˚ ) Inside– inside (%) Surface– surface (%) Nonsurface ionic (%) Hyperthermophile Pyrococcus woesei 100–103 a-Amylase 1mxd 100 24.2 0 112 34.2 30.3 17.1 46.4 0.0 [1,32,50,51] Thermophile Thermus aquaticus 70–75 Amylomaltase 1esw 16 6.6 2.60 ND 38.0 51.8 13.7 50.8 0.0 [1,52] Bacillus stearothermophilus 65–69 a-Amylase 1hvx 33 12.7 1.92 t 1 ⁄ 2 ,50 min (90 °C) 33.0 25.8 9.9 53.1 0.0 [53,54] Mesophilic organisms Room Alkalophilic Bacullus sp.707 temperature G 6 -producing amylase 1wp6 30 12.3 1.97 ND 32.6 28.9 12.6 41.7 0.0 [55] Aspergillus oryzae a-Amylase 6taa 21 10.2 2.14 62 32.3 32.5 9.1 48.6 0.4 [32,56] Plant (Barley) a-Amylase 1amy 32 13.3 1.41 ND 34.5 42.4 10.5 47.7 0.0 [57] Human a-Amylase 1b2y 18 10.0 2.52 67.4 33.3 30.6 10.5 47.4 0.0 [32,58] a The homology (%), Z score and rmsd values against the AMPw molecule were retrieved by protein structure matching in a macromolecular structure database (EMBL-EBI) (http:// www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver). I. Matsui and K. Harata Strucural elememts providing hyperthermostability FEBS Journal 274 (2007) 4012–4022 ª 2007 The Authors Journal compilation ª 2007 FEBS 4019 and percentage of polar contact on the surface did not show a significant trend from the mesophilic to the hyperthermophilic AM (Table 4). Conclusion As shown in Tables 2–4, the most thermostable enzymes from Pyrococcus species (e.g. ArATPh, BGPh, and AMPw) belonging to three different pro- tein families, have the highest rate of buried polar con- tacts compared to that of their mosophilic and thermophilic counterparts. In addition, ArATPh has a much higher rate of buried ion pairs than ATs from other species. Recent surveys on the exposure of ioniz- able groups to solvent [59], ion pairs [60], and the des- olvation energy of these residues [61] using the protein structure database, show that more than 30% of the ionizable residues are fully or partially buried and ion- ized in the internal of the molecule [62]. Buried ionized residues appear to be more conserved than those on the surface [62]. Here, the dominant roles of the buried polar contacts and ion pairs were reviewed by distin- guishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for pro- tein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. Although more abundant data for the structure ⁄ stability relationships of various proteins other than the representatives, AT, BG and AM, if available, should be considered, the results reported suggest strategies for improving the thermostability of enzymes by modulating the internal polar contacts and ion pairs. Acknowledgements We thank Hideshi Yokoyama at University of Shi- zuoka, School of Pharmaceutical Science, and Eriko Matsui for their valuable advice and discussion. 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MINIREVIEW Implication for buried polar contacts and ion pairs in hyperthermostable enzymes Ikuo Matsui and Kazuaki Harata Biological Information Research. essen- tial roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed