Effects of salt on the kinetics and thermodynamic stability of endonuclease I from Vibrio salmonicida and Vibrio cholerae Laila Niiranen1, Bjørn Altermark2, Bjørn O Brandsdal2, Hanna-Kirsti S Leiros2, Ronny Helland2, ˚ Arne O Smalas2 and Nils P Willassen1 Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science, University of Tromsø, Norway Keywords endonuclease I; kinetics; salt adaptation; thermodynamic stability; Vibrio Correspondence N P Willassen, Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway Fax: +47 776 453 50 Tel: +47 776 446 51 E-mail: nilspw@fagmed.uit.no (Received 24 October 2007, revised 14 December 2007, accepted February 2008) doi:10.1111/j.1742-4658.2008.06317.x Adaptation to extreme environments affects the stability and catalytic efficiency of enzymes, often endowing them with great industrial potential We compared the environmental adaptation of the secreted endonuclease I from the cold-adapted marine fish pathogen Vibrio salmonicida (VsEndA) and the human pathogen Vibrio cholerae (VcEndA) Kinetic analysis showed that VsEndA displayed unique halotolerance It retained a considerable amount of activity from low concentrations to at least 0.6 m NaCl, and was adapted to work at higher salt concentrations than VcEndA by maintaining a low Km value and increasing kcat In differential scanning calorimetry, salt stabilized both enzymes, but the effect on the calorimetric enthalpy and cooperativity of unfolding was larger for VsEndA, indicating salt dependence Mutation of DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters The VsEndA Q69N mutation also increased the Tm value, whereas other mutations affected mainly DHcal The determined crystal structure of VcEndA N69Q revealed the loss of one hydrogen bond present in native VcEndA, but also the formation of a new hydrogen bond involving residue 69 that could possibly explain the similar Tm values for native and N69Qmutated VcEndA Structural analysis suggested that the stability, catalytic efficiency and salt tolerance of EndA were controlled by small changes in the hydrogen bonding networks and surface electrostatic potential Our results indicate that endonuclease I adaptation is closely coupled to the conditions of the habitats of natural Vibrio, with VsEndA displaying a remarkable salt tolerance unique amongst the endonucleases characterized so far Extracellular and periplasmic enzymes of marine organisms are exposed to environments in which large variations in temperature and salinity can occur Such conditions require the proteins to fold effectively and maintain their stability in spite of the stresses they face [1] At the same time, enzymatic activity is dependent on fine-tuned structural flexibi- lity [2] How enzymes cope with these contradicting demands? The study of the structural and functional adaptation of proteins from extremophilic organisms is an active research area, and several interesting observations of the adaptive mechanisms have been made The extreme temperature stability of thermophilic Abbreviations DSC, differential scanning calorimetry; EndA, endonuclease I; VcEndA, Vibrio cholerae endonuclease I; VsEndA, Vibrio salmonicida endonuclease I; Vvn, Vibrio vulnificus endonuclease I FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1593 Effects of salt on Vibrio endonucleases L Niiranen et al proteins is thought to be a result of extensive intramolecular networks and compact packing that restrict their flexibility [3,4] Psychrophilic enzymes, in contrast, are thermolabile and have been hypothesized to use increased flexibility to cope with the increased viscosity and decreased thermal vibrations at low temperature [5,6] They may also use local flexibility to maintain a functional active site, whilst separate more rigid domains confer stability to their structure [7] Compared with non-halophiles, halophilic proteins have an excess of acidic amino acid residues that create a negative surface potential and a protective hydrated ion network [1,8] The charged surface is, however, destabilizing, especially at low salt concentrations [9], and most halophilic proteins are inactivated at NaCl or KCl concentrations below m [1] The structural basis of salt-tolerant activity remains to be elucidated, although electrostatic interactions have been implicated [10] The salinity of seawater is significantly lower than that of extreme halophile habitats, so that only milder forms of adaptation may be necessary for periplasmic and extracellular proteins of marine organisms The functional characterization of extremophilic proteins has so far focused on the obvious, i.e the effects of temperature on thermophilic and psychrophilic proteins, and salinity on halophilic proteins However, many of the environments in which extremophiles thrive are extreme with respect to more than just one parameter For example, in the field of psychrophile research, the majority of enzymes studied so far have been extracellular and of marine origin [7], which poses a problem when conclusions are to be drawn about the mechanisms of cold adaptation Are the observed adjustments a result of true adaptation to low temperature, or a combination of cold and salt adaptation? Choosing non-marine (freshwater) psychrophiles as study targets has been proposed as a solution [7] The interplay between the two types of adaptation is, however, interesting in itself, and it is possible to design experiments in a manner that facilitates the separation of the two effects The first steps towards this approach have been taken In a comparative study of a marine psychrophilic and an estuarine mesophilic endonuclease I (EndA, EC 3.1.30) [11], the different salt optima of the enzymes were taken into consideration when the temperature-dependent enzymatic properties were characterized In the discussion, the authors stressed the importance of performing measurements in buffers that were as physiological as possible Similar to psychrophilic EndA, marine carrageenase was found to display an activity optimum around the salt concentration of seawater [12] It 1594 appears that the choice of buffer and the determination of the salt dependence of the activity are important in comparative experiments on extracellular enzymes EndA is a periplasmic or extracellular sugar nonspecific endonuclease Its physiological function is not known, but it has been proposed to be involved in the prevention of the uptake of foreign DNA, the degradation of intestinal mucus to facilitate colonization, and the provision of nucleotides for the cells [13] Although EndA has been isolated from many pathogenic bacteria, it does not appear to be involved in virulence [13– 15] It may, however, affect the bacterial survival rate through the degradation of neutrophil extracellular traps in mammals, and possibly also in fish [16,17] The structures of three Vibrio endonucleases, V salmonicida (VsEndA) [18], V cholerae (VcEndA) [19] and V vulnificus (Vvn) [20], are available, and also the structure for Vvn bound to bp and 16 bp dsDNA [20,21] A reaction mechanism has been proposed based on the protein–DNA complex structure [20] Sequence identity between mature Vvn and VcEndA is 75% and between Vvn and VsEndA is 74% The structural fold and the active site containing the catalytically important His80 are identical in all three structures Temperature adaptation has not been found to affect the reaction mechanism of any homologous enzymes studied so far [22] The thermal adaptation of VsEndA and VcEndA has been studied previously, revealing that VsEndA has a higher kcat value at 5–37 °C and is less thermostable than VcEndA [11] The shape-complementary surface of Vvn contacts the DNA only at the backbone phosphate groups [20] Comparisons of Vvn, VsEndA and VcEndA have revealed that most of the charged residues in the binding cleft are conserved [18] The exceptions to this are two interesting regions with high-temperature B-factors pointed out by Altermark et al [18] The first is loop 51–54 which contains two more positive charges in VsEndA than in VcEndA, but is unlikely to contact DNA The second is residues 69 and 71 which participate in the formation of the substrate binding site These residues are Gln and Lys, respectively, in VsEndA, but both are replaced by Asn in VcEndA Intuitively, such changes in charge and steric effects may alter substrate binding and salt sensitivity In this study, the effect of NaCl concentration on the kinetic constants and thermodynamic stability of VsEndA and VcEndA was investigated In addition, the effects of reciprocal mutations of two non-conserved DNA binding site residues (VsEndA, Q69N and K71N; VcEndA, N69Q and N71K) on the kinetics, thermostability and salt dependence of these enzymes FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS L Niiranen et al Effects of salt on Vibrio endonucleases were examined Although salt stabilizes both native enzymes equally, VsEndA is adapted to retain activity at much higher salt concentrations than VcEndA The relationship between these observations and the structure determined for the VcEndA N69Q variant, as well as the previously published native EndA structures, is discussed A thorough decomposition of the thermodynamic data, together with mutational and structural investigations, was used to gain an insight into halotolerant adaptation Results Protein production and thermal stability The VsEndA variants Q69N and K71N and the VcEndA variants N69Q and N71K were expressed in a soluble form at levels comparable with the native endonucleases The mutations did not change the purification properties of the enzymes The effect of NaCl on the thermal stability of VsEndA and VcEndA was investigated by performing differential scanning calorimetry (DSC) scans in the presence of different salt concentrations The thermal stabilities of the mutated enzymes were determined at a single salt concentration (0.175 m for VcEndA and 0.425 m for VsEndA variants) chosen on the basis of the optimal activity conditions of the native enzymes [11] The denaturation peaks were symmetrical, except for some exothermic distortion of the thermograms after the denaturation peak, especially in the VcEndA sample with 0.050 m NaCl Visible aggregation was present in most samples after the thermal scan, and refolding experiments were not pursued As shown in Fig 1, NaCl stabilized both VsEndA and VcEndA by increasing the Tm value Salt also affected the shape of the thermograms, making the denaturation peaks narrower and sharper with increasing salt concentration DSC of VsEndA in 0.050 m NaCl was not performed because of sample instability The symmetrical shape of the thermograms suggests that the transition proceeds via a single transition state The increases in Tm from 0.175 to m NaCl were 10.1 and 9.0 °C for VsEndA and VcEndA, respectively (Table 1) The DHcal values increased with salt concentration, except for VcEndA above 0.425 m NaCl, although DHeff also increased in this case VsEndA Q69N showed a higher Tm value, but DHcal was unchanged All other mutants showed Tm values comparable with the native enzyme, but a lower DHcal The accordance between DHcal and the model-dependent van’t Hoff enthalpy (DHeff) was best at moderate Fig Denaturation heat capacity curves of the native and mutant VsEndA (top) and VcEndA (bottom) Differential scanning calorimetry profiles were recorded at a scan rate of °CỈmin)1 in a buffer containing 0.175, 0.425 and 1.00 M NaCl for native VsEndA, and also 0.050 M NaCl for native VcEndA For VsEndA and VcEndA mutants, 0.425 and 0.175 M NaCl, respectively, were used Thermograms were baseline-subtracted and normalized for protein concentration salt concentrations, decreasing at high extreme concentrations and for the mutants The denaturation heat capacity increment could not be determined because of irreversibility of unfolding Kinetics Kinetic measurements were made in 0–0.6 m NaCl Striking differences were observed in the Km and kcat values of the native endonucleases (Fig 2) The Km value for VcEndA increased steeply at salt concentrations above 0.25 m An equivalent increase was seen for VsEndA above 0.50 m NaCl The kcat values also showed the same salinity optima: 0.25 m for VcEndA and 0.50 m for VsEndA VsEndA was increasingly more efficient than VcEndA in terms of kcat ⁄ Km (Table 2) as the salt concentration increased The kcat ⁄ Km salt optima were not very different for the two FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1595 Effects of salt on Vibrio endonucleases L Niiranen et al Table Thermodynamic parameters of the thermal unfolding of VsEndA and VcEndA as a function of NaCl concentration determined by DSC NaCl (M) VsEndAa Native Q69N K71N VcEndAa Native N69Q N71K Tm (°C) DHcal (kJỈmol)1) DHeff (kJỈmol)1) DHcal ⁄ DHeff 0.175 0.425 1.000 0.425 0.425 41.8 44.8 51.9 49.0 45.3 268 351 371b 359 194 275 406 468b 445 275 0.97 0.86 0.79 0.81 0.71 0.050 0.175 0.425 1.000 0.175 0.175 48.8 52.9 56.8 61.9 52.6 53.7 324b 451 512 417 321 305 344b 478 528 560 415 385 0.94 0.94 0.97 0.74 0.77 0.79 The reciprocal mutations of the two residues participating in creating the substrate binding site affected both kinetic constants (Table 2) The variants displayed higher Km and kcat values, especially at high salt concentrations, except for the VcEndA N71K mutation which showed a decreased kcat value and a minimal effect on Km The catalytic efficiency of all variants was decreased compared with the native enzymes Interestingly, the salt optimum of VcEndA N69Q was shifted to zero salinity; both VsEndA variants were also more efficient than the native enzyme at zero salinity Structure of VcEndA N69Q a Molecular masses: VsEndA, 25 005 Da; VcEndA, 24 732 Da; VsEndA mutants, 24 645 Da; VcEndA mutants, 24 991 Da b Minimal values as a result of aggregation The crystal structure of VcEndA N69Q was deter˚ mined to 1.7 A resolution, and data collection and refinement statistics are presented in Table The electron density was well defined for most of the protein chain, and the mutated structure was similar to the ˚ native VcEndA with an rmsd of 0.20 A for main chain atoms Differences were found for Asn71 and the mutated residue 69 Electron density maps (Fig 3) showed that the orientation of these side chains was different from the native structure The side chain of Gln69 in VcEndA N69Q was rotated away from residue 72 and was unable to form the Asn69 OD1– Arg72 N hydrogen bond, which has been suggested to stabilize the 69–72 loop in VcEndA [18] Instead, Gln69 NE2 formed a hydrogen bond with Asn129 OD1 and a water-mediated hydrogen bond with Glu125 O Gln69 OE1 in VcEndA N69Q interacted through water molecules to both the side chain of Arg67 and to Glu125 O The orientation of Arg67 was slightly shifted relative to the native structure Interestingly, the side chain of Asn71 in the mutated structure was also rotated, interacting only with a symmetry related molecule with a hydrogen bond to Glu179 O and a water-mediated bond to Gln180 O Electrostatic calculations Fig Plot of the kinetic parameters Km (A) and kcat (B) for native VsEndA (d) and VcEndA (s) in 0–0.6 M NaCl The error bars represent maximum and minimum values enzymes (0.175 and 0.1 m for VsEndA and VcEndA, respectively), but the optimum was much broader for VsEndA 1596 The electrostatic surface potentials of the enzyme variants were calculated at the optimal salinity of the native enzymes (Fig 4) The effects of the mutations on the overall potentials were small, but some local changes were observed The mutations of VsEndA appeared to result in a less positively charged surface by increasing the exposure of a negatively charged patch (VsEndA Q69N, Fig 4B) or through the loss of a positive charge (VsEndA K71N, Fig 4C) VcEndA N69Q mutation (Fig 4E) led to the rotation of a neighbouring positive charge, Arg67, whereas, in FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS L Niiranen et al Effects of salt on Vibrio endonucleases Table Kinetic constants for native and mutant VsEndA and VcEndA at 0–0.6 [NaCl] (M) VsEndA Km (nM) 0.100 0.175 0.250 0.350 0.425 0.500 0.600 kcat (s)1) 0.100 0.175 0.250 0.350 0.425 0.500 0.600 kcat ⁄ Km (s)1ỈlM)1) 0.100 0.175 0.250 0.350 0.425 0.500 0.600 64.5 ± 32.0 ± 34.6 ± 49.5 ± 128 ± 186 ± 356 ± 1150 ± 2.77 ± 3.88 ± 7.35 ± 9.90 ± 15.8 ± 20.8 ± 22.4 ± 18.6 ± 42.9 122 213 200 123 112 62.8 16.1 M NaCl VcEndA N69Q VcEndA N71K VsEndA ⁄ VcEndA VsEndA Q69N VsEndA K71N VcEndA 10.1 96.5 ± 15.8 5.4 82.1 ± 10.7 5.2 93.5 ± 13.3 5.2 231 ± 35 13 377 ± 38 10 1240 ± 120 16 2540 ± 400 100 0.11 4.82 ± 0.16 0.15 7.64 ± 0.29 0.25 12.4 ± 0.5 0.25 23.8 ± 1.2 0.5 29.4 ± 1.2 0.4 43.7 ± 2.5 0.4 53.3 ± 5.8 0.9 50.0 93.1 133 103 78.0 35.1 21.0 67.5 82.5 92.9 99.3 248 975 1760 ± ± ± ± ± ± ± 14.6 12.0 12.0 15.5 18 64 280 35.6 44.1 115 440 2120 2720 ± ± ± ± ± ± 4.8 4.5 14 21 230 340 100 283 650 2890 9220 ± ± ± ± ± 10 29 30 380 1910 67.5 78.6 129 567 2170 ± ± ± ± ± 7.3 8.8 14 79 320 3.83 7.93 9.73 12.1 25.0 37.6 41.5 ± ± ± ± ± ± ± 0.12 0.34 0.33 0.5 0.6 1.3 4.2 2.40 4.05 5.75 6.39 4.43 1.95 ± ± ± ± ± ± 0.07 0.09 0.21 0.13 0.33 0.17 2.98 5.82 6.94 8.98 7.25 ± ± ± ± ± 0.08 0.22 0.15 0.88 1.33 1.62 2.88 4.18 4.68 4.86 ± ± ± ± ± 0.04 0.08 0.13 0.29 0.48 56.7 96.1 105 122 101 38.5 23.6 VcEndA N71K (Fig 4F), an increased positive surface potential was observed Discussion For marine organisms and their extracellular proteins, adaptation to environmental conditions can be assumed to be somewhat more complex than simple temperature or salt adaptation Previous studies of the two secreted endonucleases VsEndA (marine psychrophilic) and VcEndA (estuarine mesophilic) have shown that their activity is strongly dependent on temperature, but also on NaCl concentration [18] We studied how different salt concentrations and mutations affect the stability and kinetic constants of VsEndA and VcEndA, and found the effects to be striking, especially for VsEndA Thermal stability At 175 mm NaCl, the native enzymes display a difference of 11.1 °C in Tm The difference in Tm and the calorimetric enthalpy is small compared with that found for extremophilic DNA ligases [23] This confirms our previous finding that, for a psychrophilic enzyme, VsEndA has a relatively high temperature optimum and kinetic stability [11] The reason for the 67.5 91.7 50.1 14.5 2.09 0.717 29.7 20.5 10.7 3.11 0.786 24.0 36.6 32.3 8.25 2.23 1.8 0.72 0.30 0.11 0.060 0.068 1.2 0.96 1.3 1.5 3.6 11 0.64 1.3 4.2 14 59 160 small DTm may be linked to the charged residues, as the hydrophobic cores of the two enzymes are similar The extra salt bridges in the C-terminus of VcEndA and the smaller repulsion between the positively charged residues are the likely cause for the increased Tm value compared with VsEndA At concentrations less than m, salt interacts with proteins in a non-specific manner by neutralizing charges The addition of salt may lead to a decrease in intramolecular electrostatic repulsion, but an increase in the hydrophobic effect [24,25] Quantitative studies of the effects of NaCl on protein thermostability are scarce, but, in general, it has been found that there is a direct relationship between salinity and the upshift in the thermal unfolding temperature Tm [26–28] This agrees with our finding of a nearly equal increase in the Tm values of the two enzymes when salt is added, although the salt-induced increase in enthalpy is more pronounced in VsEndA than in VcEndA A salt-induced increase in Tm with a simultaneous decrease in DHcal has been proposed to result from stronger but less cooperative intramolecular interactions [29] In this context, cooperativity means that the protein structure unfolds as a single unit (one single transition), as opposed to several more or less independent units (several transitions) The increase in Tm and DHcal of EndA with increasing salt concentration FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1597 Effects of salt on Vibrio endonucleases L Niiranen et al Table X-ray data collection and crystallographic refinement statistics for the VcEndA N69Q structure Data collection X-ray source Space group ˚ Unit cell (A) ˚ Resolution (A) (highest bin) ˚ Wavelength (A) No unique reflections Multiplicity Completeness (%) Mean ( ⁄ ) R-sym (%)a ˚ Wilson B-factor (A2) Refinement PDB entry ˚ Resolution (A) R-factor (all reflections) (%) R-free (%)b No of atoms No of water molecules No of other molecules ˚ rmsd bond lengths (A) rmsd bond angles (deg) ˚ Average B-factor (A2) All atoms Protein Water molecules ⁄ Mg2+ ⁄ Cl) Ramachandran plot Most favoured regions (%) Additionally allowed regions (%) Generously allowed regions (%) In-house rotation anode P 212121 a = 40.26, b = 64.41, c = 75.64 25.00-1.70 (1.79-1.70) 1.54180 22 098 2.9 (2.8) 99.3 (99.1) 12.8 (2.2) 6.5 (35.6) 20.5 2VND 15.00-1.70 19.7 25.9 1928 223 Mg2+, Cl) 0.017 1.520 Kinetics 17.1 16.1 24.4 ⁄ 22.7 ⁄ 11.1 93.9 5.5 0.6 P P P P R-sym = ( h I | Ii(h) – |) ⁄ ( h I I(h)), where Ii(h) is the ith measurement of reflection h and is the weighted mean of all measurements of h b 5% of the reflections were used in the R-free calculations a could therefore be interpreted, conversely, as increased cooperativity of unfolding and a more compact structure as a result of stronger intramolecular interactions These salt-induced effects on DHcal and DHeff are stronger in VsEndA, possibly because of the increased number of solvent-exposed charged and hydrophobic residues relative to VcEndA, as found when viewing the molecular surfaces and their amino acid properties This indicates a certain degree of salt dependence of VsEndA stability, but is in disagreement with the observation that the Tm values for both enzymes are equally affected by salt addition It has been suggested that salt stabilizes halophilic proteins to a greater extent than non-halophilic proteins, and that halophilic proteins are destabilized by low salt concentrations [9] Both effects may originate from the characteristic high negative surface potential of halophilic proteins and increased solvent ion binding [8,30,31] The equal 1598 increase in Tm of VsEndA and VcEndA may suggest that VsEndA does not have any specific ion binding sites on its surface relative to VcEndA, and is not halophilic The more highly charged surface of VsEndA may, however, constitute a more cooperative solvent ion binding network, which makes it possible for the enzyme to better tolerate fluctuations in salt concentration The observed general decrease in DHcal ⁄ DHeff with increasing salt concentration may imply that the theoretical model used is unable to tackle the increased cooperativity of unfolding, but may also be explained by an increase in the degree of irreversible unfolding (Table 1) The reversibility of thermal unfolding of a halophilic b-lactamase has been found to be inversely dependent on salt concentration, and has been proposed to be caused by the salting-out effect of NaCl [28] NaCl can neutralize the surface charges of unfolded proteins and facilitate aggregation The release of coordinated ions and water molecules from the solvation shells of enzymes and substrates provides a positive entropic effect that drives substrate binding This effect is dependent on both temperature and salt concentration [32,33] At elevated salt concentrations or low temperatures, the gain in entropy on release of ions is reduced and substrate binding is therefore weaker [33] This makes binding of highly charged DNA very challenging for marine enzymes DNA binding to non-halophilic proteins has been found to be inversely dependent on salt concentration [32,34], whereas the binding efficiency of halophilic proteins appears to actually increase with increasing salt concentration [35,36] A halophilic nuclease from Micrococcus varians [37] with maximal activity in 3–4 m NaCl displays an excess of acidic residues characteristic of many halophilic enzymes It is possible that this enzyme has a binding mechanism involving counterion uptake, similar to that proposed for the halophilic Pyrococcus woesei TATA-box binding protein [36] Contrary to these halophilic proteins, VsEndA displays an excess of basic residues contacting the negatively charged substrate, and the Km value increases with increasing salt concentration, although this occurs at a much higher salinity than for VcEndA In our previous study of EndA temperature adaptation, the more positively charged surface of VsEndA was considered not to decrease the Km value relative to VcEndA [11] These measurements were made at the respective optimal salt concentrations of the enzymes, where the Km values were found to be of the FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS L Niiranen et al Effects of salt on Vibrio endonucleases Fig (A) Electron density (2Fo – Fc at 1r contoured in blue) and omit (Fo – Fc at 3r contoured in green) maps illustrating the orientation of Asn71 and the N69Q mutation in the VcEndA N69Q structure (B) Superposition of the VcEndA N69Q mutant (red), native VcEndA (blue) and VsEndA (green) structures (C) A partial sequence alignment of VsEndA and VcEndA The asterisks indicate the non-conserved residues selected for mutagenesis, and the plus sign denotes the catalytically important His80 Sequence numbering follows that of Vvn [20] same magnitude This can be explained by the similar or slightly lower electrostatic surface potential of VsEndA compared with VcEndA at the respective optimal salinities (Fig 4A,D) The results of the present study show that the Km values are strongly affected by the NaCl concentration, similar to the surface charge of the enzyme The higher positive charge of VsEndA therefore decreases Km, but this is a method of coping with the charge shielding of buffer solutes rather than low temperatures The higher charge may allow VsEndA to retain sufficient charge, even at relatively high salinity, to enable tight substrate binding, contrary to VcEndA The salt adaptation of kinetic constants as striking as that observed in the present study has not been presented previously Only two comparative studies of the salt-dependent kinetics of a non-halophilic and a saltadapted enzyme have been published to date In the comparison of halotolerant Dunaliella salina carbonic anhydrases dCA I and dCA II and the human homologue in 0–0.5 m NaCl, the largest differences were found in the Km values [10,38] Similar to our results, the halotolerant enzymes retained a low Km, whereas the Km value of the non-halophilic enzyme increased considerably with the addition of salt These results imply that Km salt tolerance is a feature typical to halotolerance The role of kcat is less clear Both Bageshwar et al [38] and Premkumar et al [10] found kcat to be increased only slightly by salt, whereas we observed a large effect for kcat for both VsEndA and VcEndA at high salinity The higher catalytic rate may reflect the dependence of kcat on the substrate binding and dissociation rate constants, as found in the cold adaptation of cod trypsin [39], or, in the case of VsEndA, may be linked in some way to cold adaptation, where an increase in kcat is a typical mechanism [7] A high kcat value may also be a feature of salt tolerance, but more studies on halotolerant enzymes are required to verify this The addition of salt may cause the EndA substrate binding cleft to reach a more optimal configuration for enzyme catalysis, thereby affecting kcat The DSC thermograms indicate that salt constricts the structural fluctuations of the enzyme At a certain concentration, these fluctuations may become optimal for enzymatic turnover, whereas, at salt concentrations above the optimum, the structure becomes too rigid and will function less optimally If the stabilizing effect of salt is caused mainly by the weakening FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1599 Effects of salt on Vibrio endonucleases L Niiranen et al A D B E C F Fig Electrostatic surface potentials in the DNA binding groove of VsEndA with a modelled DNA (A), VsEndA Q69N (B) and VsEndA K71N (C) all in 0.425 M NaCl, and VcEndA (D), VcEndA N69Q (E) and VcEndA N71K (F) all in 0.175 M NaCl The black arrows show the mutated residues The surface potential is coloured from )10 kT ⁄ q (red) to 10 kT ⁄ q (blue) of repulsive charges, it is reasonable to imagine that VsEndA must be screened by a higher salt concentration than VcEndA to be able to function optimally Effects of mutations The point mutations are not in the immediate vicinity of the active site situated at the bottom of the positively charged pocket, but are still likely to affect the shape, stability and charge of the DNA binding site (Fig 4) The Asn69 side chain in VcEndA forms a 1600 hydrogen bond to Arg72 N, which may stabilize this loop region relative to Vvn and VsEndA [19], whereas the hydrogen bond observed in the VcEndA N69Q structure (Gln69 to Asn129) stabilizes other regions The characterization of the VcEndA N69Q mutant (Table 2) shows higher Km and kcat values compared with native VcEndA The lost hydrogen bond (from 69 to 72) in VcEndA N69Q may increase the flexibility of the 69–72 loop, possibly explaining the decreased binding affinity and increased catalytic rate In addition, the shape of the DNA binding pocket in VcEndA FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS L Niiranen et al N69Q is slightly altered as both residues 71 and 69 are moved in the crystal structure (Fig 3), and the current orientation of Gln69 is different from the Vvn–DNA structure (Fig 3B) and very close to a modelled DNA backbone, possibly explaining the higher Km values (Table 2) Gln69 in VsEndA is poorly defined in the native crystal structure, and the characterization of the VsEndA Q69N mutant (Table 2) reveals poorer DNA binding and an increased kcat with a maximum at 0.5 m NaCl The Gln69 side chain in the Vvn–DNA ˚ structure (PDB 1OUP) is less than 3.2 A from the DNA backbone, and mutation to the shorter Asn in VsEndA Q69N may prevent the formation of favourable DNA–enzyme interactions, and lead to the higher Km values observed In addition, the Asn69 to Arg72 hydrogen bond lost in the VcEndA N69Q structure may be formed in VsEndA Q69N, although this should be verified by structural studies This additional hydrogen bond may explain the increased stability of the VsEndA Q69N mutant, and the subsequent decrease in flexibility may further impair substrate binding and contribute to the higher Km values The introduction or removal of a positively charged residue (N71K and K71N) has a large effect on the electrostatic surface potential (Fig 4) However, the VcEndA N71K mutant has a binding affinity and turnover comparable with the native VcEndA Being more distal from DNA, as observed in the Vvn–DNA structure, residue 71 may have less influence on DNA binding than residue 69 Interestingly, kcat starts to decrease when the salt concentration exceeds 0.25 m for both native VcEndA and VcEndA N69Q, but this is not observed for the VcEndA N71K variant The VsEndA K71N mutant shows poorer DNA binding and increased kcat compared with VsEndA, indicating that the positive charge is more important for DNA binding in VsEndA than in VcEndA No side chain contacts are seen for residue 71 in the native structures or models of the mutants As the longer side chain of lysine has more rotamers, the N71K substitution in VcEndA may stabilize the structure by increasing the rotational entropy, whilst retaining the backbone interactions An increase of °C is observed for the Tm value of this mutant In VsEndA K71N, both DHcal and the cooperativity of unfolding are decreased, possibly indicating changes in the hydrogen bonding networks The increase in Km may be the result of a slightly enlarged binding site or less positive charge Indeed, the changes seen in the electrostatic surface potential of each of the mutants (Fig 4) match surprisingly well with their kinetic results Both VsEndA mutants and the VcEndA N69Q mutant show more dispersed or less positive charge, and, accord- Effects of salt on Vibrio endonucleases ingly, display higher and more salt-sensitive Km values VcEndA N71K does not display a lower Km value, but one similar to the native enzyme, in spite of the acquisition of an additional positive charge, possibly because of other effects caused by the mutation Even minor changes in protein structure, such as single amino acid replacements, can induce a significant change in the cooperativity of unfolding, and be detected as changes in the effective (van’t Hoff) enthalpy [40] In the DSC experiments, only the VsEndA Q69N mutation had the expected effect, increasing the stability via both Tm and the cooperativity of unfolding, although the DHcal value was comparable with that of the native enzyme Whether or not local changes are reflected in the global hydrogen bonding networks, and how widespread are their effects, cannot be discerned from the native and mutant crystal structures Effects on hydrogen bonding networks may change the electron density distribution around and in the active site and, together with small conformational alterations, may speed up the rate-limiting step of hydrolysis Such long-range effects have been proposed to be the cause of more mesophilic-like kinetic behaviour in psychrophilic a-amylase, where single amino acid mutations were introduced outside the catalytic cleft [22,41] The identity of the rate-limiting step in the endonuclease reaction mechanism is not known However, the high catalytic efficiency of both VsEndA and VcEndA (kcat ⁄ Km in the region of 108 s)1Ỉm)1) shows that the reaction is nearly diffusion controlled, suggesting that the rate-limiting step is either substrate binding or dissociation As all mutations affect Km, especially at high salt concentrations, the optimization and salt tolerance of binding interactions are most probably hampered by the mutations by electrostatic, steric or flexibility effects The less tight binding of DNA may enable the enzymes to release the products more easily, thus leading to the observed increase in the kcat values of three of the variants Similarly, the seven-fold higher kcat value of the hyperactive variant of Escherichia coli dihydrofolate reductase, compared with the wild-type enzyme, has been suggested to result from increased flexibility and size of the substrate binding cleft, leading to an increased product dissociation constant [42] Conclusions The experiments conducted in this study show that the secreted endonuclease VsEndA from the marine psychrophilic V salmonicida is remarkably salt tolerant and therefore unique amongst the endonucleases characterized so far Salt has striking effects on the kinetic FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1601 Effects of salt on Vibrio endonucleases L Niiranen et al constants of VsEndA, and the high positive charge of VsEndA is considered to be essential in counteracting the charge shielding of buffer solutes and maintaining a low Km at high salinity It is possible that Km salt tolerance will emerge as a general feature for halotolerant proteins The role of the high kcat value observed for VsEndA is less clear, and more studies on halotolerant enzymes are required to elucidate this further The salt-induced increase in enthalpy and cooperativity of unfolding is more pronounced in VsEndA This effect indicates the formation of a more compact structure through the strengthening of intramolecular interactions or the weakening of intramolecular repulsive forces, and the salt dependence of VsEndA stability The higher positive electrostatic surface potential of VsEndA compared with VcEndA plays a key role in adaptation On the whole, the characteristics of VsEndA and VcEndA illustrate the fine-tuned adaptation to their natural environments Materials and methods Site-directed mutagenesis and plasmid purification Residue targets for mutagenesis were selected on the basis of the sequence and structural alignments of Vvn, VsEndA and VcEndA The selected residues 69 and 71 were nonconserved between VsEndA and VcEndA, located in the DNA binding region and close to the active site Site-directed mutagenesis was performed using a QuikChange SiteDirected Mutagenesis Kit (Stratagene, Cedar Creek, TX, USA), as described in the manual The oligonucleotides were synthesized by Sigma-Aldrich (St Louis, MO, USA) Mutated plasmids were transformed into E coli TOP10 cells (Invitrogen, Carlsbad, CA, USA), and plasmid extraction was performed using QIAprep minipreps (Qiagen, Hilden, Germany) or the alkaline lysis method [43] Expression and purification The expression and purification of recombinant VsEndA and VcEndA native enzymes and mutants were performed as described previously [11] with a few modifications Cells were cultured in either shake-culture flasks or a Techfors S fermenter (Infors, Bottmingen, Switzerland) The culture temperature was kept at 37 °C until glucose was depleted, after which the temperature was adjusted to 22 °C before expression was induced The cells were harvested when they reached the stationary phase and were collected by centrifugation For periplasmic fractionation, the cells were resuspended in a : 10 culture volume of fractionation buffer, and incubated on ice for 1–1.5 h before the supernatant was collected 1602 Enzyme assay Enzyme activity measurements were assayed in triplicate at 23 °C in 75 mm Tris ⁄ HCl, pH 8.0 and pH 8.5 (VcEndA and VsEndA, respectively), mm MgCl2 and 0–0.6 m NaCl Eight different concentrations (12–1470 nm) of DNaseAlert substrate (DNaseAlertÔ QC System Kit; Ambion, Austin, TX, USA) were used for the kinetic measurements, and 200 nm substrate for the other activity measurements The total reaction volume was 100 lL and reactions were started by the addition of 10 lL of enzyme diluted in reaction buffer Protein LoBind tubes from Eppendorf (Hamburg, Germany) were used for enzyme dilutions because of the sticky nature of the enzyme The detailed assay procedure is described elsewhere [11] sigmaplot software (Systat Software, San Jose, CA, USA) was used for data analysis, and Vmax and Km values were calculated by fitting the velocity data to the Michaelis–Menten equation Differential scanning calorimetry Differential scanning calorimetry experiments were conducted on a Nano-Differential Scanning Calorimeter III, model CSC6300 (Calorimetry Sciences Corporation, Lindon, UT, USA) Preparations of the native enzymes were first filtered with a 0.45 lm Spin-X centrifuge tube filter (Corning, Corning, NY, USA), and then dialysed overnight at °C against L of dialysis buffer (50 mm Hepes, mm MgCl2, pH 8.0) containing 0.050, 0.175, 0.425 or 1.00 m NaCl Slide-A-Lyzer dialysis discs from Pierce (Rockford, IL, USA) with a kDa cut-off were used The protein concentration of the dialysed enzyme solution was determined using BioRad Protein Assay Dye Reagent Concentrate (BioRad, Hercules, CA, USA) with bovine serum albumin (Sigma) as standard The dialysates were used as blank references in DSC runs Reference buffers and samples were carefully degassed before loading into the DSC cells The scans were performed at a constant pressure of 304 kPa in the range 15–75 °C or 20–80 °C with a heating rate of °CỈmin)1 Thermograms were analysed according to a single non-two-state transition model in which Tm, DHcal and DHeff were fitted independently using cpcalc software (Calorimetry Sciences Corporation) Crystallization, data collection and structure determination The mutant VcEndA N69Q was crystallized in similar conditions as native VcEndA [19] using the hanging drop vapour-diffusion technique at room temperature with 6.2 mgỈmL)1 of protein in 50 mm Tris ⁄ HCl pH 8.0, mm MgCl2 and 0.6 m NaCl Drops were made by mixing lL of protein with lL of reservoir solution consisting of 0.1 m sodium acetate, 0.3 m ammonium acetate, 10 mm magnesium sulphate and 26% PEG8000 Crystals of about FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS L Niiranen et al 500 · 200 · 20 lm3 were transferred to cryoprotectant solution with 30% PEG8000, 15% glycerol and the other reservoir additives, and flash-cooled in liquid nitrogen Data were collected at the in-house MicroMax-007 HF rotating anode from Rigaku (Osaka, Japan) with an R-AXIS IV detector, a 60 s exposure time per image and 0.5° oscillation, and a total of 78° of data were used in the final data set The data were integrated with the program mosflm [44], scaled with scala, and the structure factors obtained with truncate in the ccp4 program suite [45] The structure was solved by molecular replacement using the program molrep [46] in ccp4 and the structure of native VcEndA (PDB code 2G7E) as a search model The structure was refined in refmac5 [47] interspersed with rounds of manual model building in O [48] based on rA-weighted 2Fo – Fc and Fo – Fc electron density maps The final model was validated using procheck [49] Molecular modelling and electrostatic calculations Continuum electrostatic calculations were carried out using the delphi program package [50,51] The parse3 set of atomic radii [52], together with formal charges, was used in all calculations The electrostatics were determined using the linear Poisson–Boltzmann equation and a three-dimensional grid with a size of 165 · 165 · 165 Stepwise focusing was used to increase the accuracy [53] Initially, a rough grid was calculated with Coulombic boundary conditions, and the resulting grid was adopted as the boundary condition for one further focused calculation The protein molecules occupied 90% of the box in the final calculations The molecular surface was calculated using a solvent probe of ˚ 1.4 A The solvent was described using a dielectric constant of 80, whereas the protein was treated with a dielectric constant of The 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Struct-Theochem 729, 11–18 FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1605 ... stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA Possible thermodynamic origins of the ‘‘glutamate effect’’ on protein–DNA... function optimally Effects of mutations The point mutations are not in the immediate vicinity of the active site situated at the bottom of the positively charged pocket, but are still likely... ⁄ Km in the region of 108 s)1Ỉm)1) shows that the reaction is nearly diffusion controlled, suggesting that the rate-limiting step is either substrate binding or dissociation As all mutations affect