Báo cáo khoa học: Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases pdf

Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases Kevin L Epting1, Claire Vieille2, J Gregory Zeikus2 and Robert M Kelly1 Department of Chemical Engineering, North Carolina State University, Raleigh, NC, USA Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA Keywords inactivation kinetics; metal cofactors; thermostability; xylose isomerases Correspondence R M Kelly, Department of Chemical Engineering, North Carolina State University, Raleigh, NC 27695–7905 Fax: +1 919 515 3465 Tel: +1 919 515 6396 E-mail: rmkelly@eos.ncsu.edu (Received 27 November 2004, accepted 20 January 2005) doi:10.1111/j.1742-4658.2005.04577.x The effects of divalent metal cations on structural thermostability and the inactivation kinetics of homologous class II d-xylose isomerases (XI; EC 5.3.1.5) from mesophilic (Escherichia coli and Bacillus licheniformis), thermophilic (Thermoanaerobacterium thermosulfurigenes), and hyperthermophilic (Thermotoga neapolitana) bacteria were examined Unlike the three less thermophilic XIs that were substantially structurally stabilized in the presence of Co2+ or Mn2+ (and Mg2+ to a lesser extent), the melting temperature [(Tm) %100 °C] of T neapolitana XI (TNXI) varied little in the presence or absence of a single type of metal In the presence of any two of these metals, TNXI exhibited a second melting transition between 110 °C and 114 °C TNXI kinetic inactivation, which was non-first order, could be modeled as a two-step sequential process TNXI inactivation in the presence of mm metal at 99–100 °C was slowest in the presence of Mn2+ [half-life (t1 ⁄ 2) of 84 min], compared to Co2+ (t1 ⁄ of 14 min) and Mg2+ (t1 ⁄ of min) While adding Co2+ to Mg2+ increased TNXI’s t1 ⁄ at 99–100 °C from to 7.5 min, TNXI showed no significant activity at temperatures above the first melting transition The results reported here suggest that, unlike the other class II XIs examined, single metals are required for TNXI activity, but are not essential for its structural thermostability The structural form corresponding to the second melting transition of TNXI in the presence of two metals is not known, but likely results from cooperative interactions between dissimilar metals in the two metal binding sites Enzymes from hyperthermophiles are intrinsically thermostable and thermoactive, with optimal temperatures of activity often in excess of 100 °C [1] Studies focusing on the molecular basis for the thermostability of these enzymes have revealed an array of subtle contributing factors, including larger hydrogen bonding and ion pairing networks, additional inter-subunit interactions in oligomeric proteins, decreased labile amino acid content, lower surface-to-volume ratios, and smaller loops [2–4] These factors can often be identified by comparing the structures of homologous enzymes with varying degrees of thermostability [5] However, the extent to which these individual factors contribute to thermostability is highly specific to particular enzymes [6] A potential contributing factor to enhanced thermostability that has not been examined in much detail is the role that metals play in stabilizing and activating enzymes from hyperthermophiles in comparison with their less thermophilic counterparts Xylose isomerase Abbreviations TIM, triosephosphate isomerase; XI, xylose isomerase 1454 FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS K L Epting et al (XI) (d-xylose ketol isomerase, EC 5.3.1.5) is an excellent model system to consider in this regard, as divalent metal cations are important for both stability and activity of all known XIs [7] XI converts d-xylose to d-xylulose in vivo, but it also converts d-glucose to d-fructose in vitro [8], hence its use for the commercial production of high fructose corn syrup [9,10] An intracellular enzyme, XI is found in a number of bacteria that can grow on xylose [11], as well as in fungi [12,13], and plants [14] XIs can be divided into two groups based on sequence comparisons: class I and class II [15] Class I XIs contain % 390 amino acids, while class II XIs typically contain around 440 amino acids and are distinguished from the class I enzymes by a 30–40 amino acid N-terminal insert [10] The functional role of this N-terminal insert in the class II enzymes is unknown Divalent metal cations (chosen from Co2+, Mg2+, and Mn2+) are essential for XI’s stability and activity [16–19], but their relative importance differs somewhat for class I and class II XIs [7] A number of three-dimensional structures have been solved for class I XIs [20–23] and have been shown to be essentially identical, which explains the similar biochemical and thermostability properties of these enzymes [7] Comparisons between the structures of the most thermostable class I XIs from Thermus caldophilus and Thermus thermophilus to those from less thermophilic class I XIs from Arthrobacter B3728 and Actinoplanes missouriensis revealed common thermostabilizing features: increased ion pairing, lower surfaceto-volume exposure, fewer exposed labile amino acids, and shortened loops [24] Similar comparisons among class II XIs have not been reported Class II XIs have not been studied to the same extent as class I enzymes, probably because none are currently used commercially Genes encoding several class II XIs have been cloned from mesophilic, thermophilic, and hyperthermophilic bacteria, expressed in Escherichia coli, and characterized biochemically [25–29] Although class I and class II XIs differ in their metal specificities [18], active-site structure and metal-binding residues are conserved across the two XI classes [7] In contrast to class I XIs, however, the thermostability of class II XIs is more variable No obvious differences in the enzyme structures can explain these variations in stability, although the more thermophilic class II XIs contain additional prolines and fewer thermally labile asparagine and glutamine residues [26,30] The crystal structures of class I and class II XIs show that these enzymes are typically homodimers or homotetramers, consisting of a triosephosphate isomerase (TIM) barrel connected to a C-terminal loop FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS Thermostability of class II xylose isomerases [21,31] Each monomer contains an active site that features two distinct metal binding sites Metal M1 is coordinated by four carboxylate groups, while metal M2 is coordinated by one imidazole and three carboxylate groups [32] Metals M1 and M2 were initially called structural and catalytic metals, respectively, due to the fact that the M1 site remained geometrically unchanged during catalysis, while the M2 site changed upon substrate binding [33] Subsequent studies have shown, however, that both metals are needed for catalysis [34] The binding affinity appears to differ for the two sites [17,35,36], and it varies with pH and the type of metal [37] Binding affinity, though, varies in the same way in both enzyme classes, with Mn2+ > Co2+ > Mg2+ [19] Metal specificity also depends on both the nature of the substrate (i.e glucose or xylose) and on the enzyme class [26] With glucose as the substrate, class I enzymes are best activated by Mg2+ [38], or, in some cases, by a combination of Mg2+ and Co2+ [11,39] While Co2+ best activates the class II enzymes for activity on glucose, Mn2+ is preferred for activity on xylose [38] However, Mn2+ or Co2+ provide superior thermal stabilization to both XI classes [16] Metal specificity appears to be related to the residues surrounding the metal binding sites Several mutant XIs with altered metal specificity were created by site-directed mutagenesis [40–42] In general, the resulting mutants had decreased specificity for all metals compared to the wild-type enzyme Given that metals are needed for both stabilizing and activating XIs, a question arises concerning the relative importance of these cofactors across functional temperature ranges This issue was examined here for class II XIs from the mesophiles E coli (ECXI) and Bacillus licheniformis (BLXI), for the moderate thermophile Thermoanaerobacterium thermosulfurigenes (TTXI), and for the hyperthermophile Thermotoga neapolitana (TNXI) Results indicate that the roles that divalent metals play in TNXI stabilization and activation differ from those in the less thermophilic enzymes Results Structural thermostability of class II XIs Four class II XIs (i.e ECXI, BLXI, TTXI, and TNXI) were investigated to determine the influence of specific divalent metal cations on thermally induced denaturation using differential scanning calorimetry (DSC) In the absence of metal (i.e apoenzymes) or in presence of a single metal at lm, all four XIs exhibited a single irreversible thermal transition (Fig 1) While the nature of the metal present significantly affected the 1455 100 Excess heat capacity (kcal/mole·K) 25 TNXI Mg2+ Apo 80 2+ Mg 2+ Mn 20 80 90 100 110 120 60 Mg2+ Apo BLXI Co2+ 20 60 70 80 90 BLXI 100 Mn2+ 150 Co2+ Apo Mg2+ 100 50 40 60 50 ECXI 50 60 70 80 90 Mg2+ Mn2+ Apo 40 Co2+ 30 20 10 40 10 Mn2+ 40 200 15 TTXI 80 Excess heat capacity (kcal/mole·K) Co Mn2+ 40 50 50 60 70 80 90 Temperature (ºC) Fig Thermal denaturation of class II xylose isomerases DSC scans of TNXI, TTXI, BLXI and ECXI were run in 50 mM Mops (pH 7.0) with no metal (apo) or in the presence (at lM) of a single metal stabilization of BLXI and TTXI relative to their apo forms, the effect was smaller for ECXI and almost negligible for TNXI (Fig 2) The melting temperatures (Tm, the temperature at the maximum of the heat capacity profile) of ECXI, BLXI, and TTXI followed the trend reported previously for class II XIs, with Mn2+ or Co2+ providing greater stabilization than 1456 2+ 20 Co2+ 60 70 Excess heat capacity (kcal/mole·K) K L Epting et al ∆T (ºC) Excess heat capacity (kcal/mole·K) Thermostability of class II xylose isomerases TTXI ECXI TNXI TNXI* Fig Effect of metals on the Tm values of class II XIs DT is the difference between Tm (enzyme in the presence of lM single metal) and Tm (apoenzyme) For TNXI*, the metal concentration was mM Mg2+ This was particularly noticeable for BLXI; Mn2+ or Co2+ increased the Tm by 20 °C more than did Mg2+ In all instances, but to varying extents, the apoenzyme melted at a lower temperature than the same enzyme containing any of the three metals (Table 1) It was interesting to note that TNXI melting curves at low metal concentrations (5 lm) led to Tm values between 96.9 °C and 97.6 °C, barely above the Tm of the apoenzyme (96.4 °C) Atomic emission spectroscopy analyses of TNXI at low metal concentrations (i.e between and 500 lm metal) suggest that both metal binding sites are not occupied in these conditions (data not shown) In the presence of excess metal (i.e mm, to ensure occupation of both metal sites), TNXI’s Tm slightly increased to 100.5 °C (Mn2+), 100.4 °C (Mg2+), and 100.0 °C (Co2+) The melting behavior of TNXI was also examined in the presence of Ni2+ and Ca2+, two divalent metals that inactivate the enzyme [11] With Tm values of 100.9 °C and 100.5 °C for mm Ni2+ and mm Table Effect of activating divalent cations on melting temperatures of class II XIs Melting temperature Tm (°C) Enzyme Apo Mg2+ Co2+ Mn2+ ECXI BLXIa TTXI TNXI TNXIb 50.8 50.3 64.1 96.4 – 56.2 53.3 83.0 97.6 100.4 56.2 73.4 86.0 97.5 100.0 57.3 73.6 86.1 96.9 100.5 a b Data from [26] Buffer containing mM metal chloride FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS Thermostability of class II xylose isomerases Excess heat capacity (kcal/mole·K) 100 100 100 80 80 60 60 40 40 20 20 70 80 90 100 110 Temperature (ºC ) Percent soluble enzyme Ca2+, respectively, both metals stabilized TNXI to the same extent as Mn2+, Mg2+, or Co2+ The biochemical and biophysical properties of some XIs have been studied in the presence of two different metals (e.g Mg2+ and Co2+) [43–45] For three of the enzymes studied here, the Tm in the presence of mm Mg2+ plus 0.5 mm Co2+ was slightly above that afforded by the most stabilizing single metal: ECXI (58.0 °C vs 57.3 °C with Mn2+), BLXI (74.4 °C vs 73.6 °C with Mn2+), and TTXI (87.0 °C vs 86.1 °C with Mn2+) In these cases, the trajectories of the melting curves were similar to those obtained in the presence of a single metal (data not shown) In contrast, TNXI’s melting curve in the presence of mm Mg2+ plus 0.5 mm Co2+ showed two transitions, around 99.5 °C and 110 °C (Fig 3) To determine whether the relative concentrations of the two metals affected TNXI melting behavior, TNXI’s melting curve was recorded in the presence of mm Mg2+ plus either or mm Co2+ In both cases, two transitions were observed (data not shown) at the same temperatures as in the presence of mm Mg2+ and 0.5 mm Co2+ In fact, all TNXI melting curves in the presence of any two of the three metals showed two transitions (Fig 3); the Mn2+ ⁄ Mg2+ and Mn2+ ⁄ Co2+ combinations showed transitions at 99.6 °C ⁄ 114.2 °C and at 100.6 °C ⁄ 112.1 °C, respectively To further investigate the basis for the two transitions, individual TNXI scans in the presence of mm Mg2+ and 0.5 mm Co2+ were stopped at 75, 90, 95, 100, 105, 110, and 115 °C (Fig 4) to view the residual soluble enzyme conformation on SDS and native Excess heat capacity (kcal/mole·K) K L Epting et al 120 Fig Soluble TNXI concentration during thermal denaturation in the presence of mM Mg2+ plus 0.5 mM Co2+ The concentration of soluble protein (% of starting concentration, h) and the thermal transitions are shown as functions of the temperature PAGE All samples showed a single band at 50 kDa on SDS ⁄ PAGE (not shown), as expected for the TNXI monomer On the native gel, however, all samples showed three distinct bands, presumably the tetramer, dimer, and monomer (Fig 5) In comparison, in the presence of a single metal, no soluble protein was present at temperatures beyond the end of the single thermal transition (not shown) All three forms of the enzyme were observed on native PAGE over the entire temperature range Despite the decrease in soluble protein concentration (Fig 4), the relative amounts of each form appear to remain the same, indicating that as the dimer dissociates into monomers, the monomers unfold and aggregate Hence, the amount of monomer in the soluble fraction remains low 80 60 40 20 70 80 90 100 Temperature (ºC) 110 120 Fig TNXI melting transitions in the presence of two different metals DSC scans were run in 50 mM Mops (pH 7.0) in the presence of mM Mg2+ ⁄ 0.5 mM Co2+ (dashed line), mM Mn2+ ⁄ 0.5 mM Co2+ (black line), or mM Mg2+ ⁄ mM Mn2+ (gray line) FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS Fig Native PAGE of soluble TNXI at different temperatures during thermal denaturation in the presence of mM Mg2+ plus 0.5 mM Co2+ Soluble fractions were loaded at 100 lg protein per lane Control: unheated TNXI in 50 mM Mops (pH 7.0) containing mM Mg2+ plus 0.5 mM Co2+ Presumed tetramer, dimer and monomer bands identified 1457 Thermostability of class II xylose isomerases K L Epting et al mm Mg2+ and 0.5 mm Co2+ (Fig 6) With a Tm of 100.7 °C, the M1 site mutant behaved like TNXI in the presence of an excess single metal Both the M2 site mutant (Tm of 95.5 °C) and the double mutant (Tm of 96.5 °C) behaved like the apo-TNXI (Tm of 96.4 °C) 140 E232K / D309K Excess heat capacity (kcal/mole·K) 120 100 80 D309K E232K 60 Kinetic inactivation 40 Previously, BLXI was shown to follow first order kinetic inactivation [26] To determine whether the two thermal transitions observed by DSC for TNXI in the presence of two metals had any relevance to this enzyme’s inactivation kinetics, the inactivation course of TNXI in the presence of various metal concentrations and metal combinations was determined Figure shows the inactivation courses of apo-TNXI and of TNXI in the presence of mm concentrations of each of the three divalent cations (Mg2+, Mn2+, or Co2+) With the exception of apo-TNXI, whose inactivation was first-order, TNXI exhibited non-first order inactivation in the presence of any single metal or combination of metals (data not shown) Previously, TNXI inactivation was proposed to proceed by a twostep, sequential mechanism [46]: 20 80 85 90 95 100 Temperature (ºC) 105 110 Fig Thermal denaturation of TNXI metal site mutants DSC scans of TNXI and its E232K, D309K, and E232K ⁄ D309K mutants in buffer A To determine if TNXI’s two melting transitions in the presence of two different metals were a result of differences in metal binding affinity in the two metal binding sites, point mutations were introduced selectively into metal sites M1 (i.e mutation E232K) and M2 (i.e mutation D309K) The double mutant (E232K ⁄ D309K) was also created In previous studies of class I XIs, the residues corresponding to E232 and D309 were mutated to lysine and the crystal structures of the mutants were determined The positive charge of the lysine’s e-amino group presumably replaced the metal, while leaving the other metal site unaffected Both mutations eliminated activity [21,31] Here, as expected, none of the three TNXI mutants were active The three mutants showed a single melting transition in the presence of Mn 2+ Relative Activity Relative Activity k2 where E is the native, fully active enzyme (relative activity of 1.0); E1 is an intermediate with lower activity (b < 1.0) than E; Ed is the inactive enzyme and k1 and k2 are the inactivation rate constants Such a mechanism can be modeled by the sum of two exponential terms, where y(t) represents the fractional residual activity [47]: Mg 2+ 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 0 20 40 60 80 100 120 20 Time (min) 40 60 80 Time (min) Co2+ Apo 1 0.8 Relative Activity Relative Activity b 1 0.6 0.4 0.2 0 1458 k1 ! E À E1 À Ed ! 20 40 60 80 100 120 140 160 180 200 Time (min) 0.8 0.6 0.4 0.2 0 20 40 60 80 100 Time (min) 120 140 Fig Model fit to TNXI kinetic inactivation in the presence and absence of activating divalent cations Model parameters and r2 values are listed in Table 106 °C (r), 104 °C (h), 102 °C (n), 99 °C (n), 96 °C (m), 90 °C (d), 87 °C (s) FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS K L Epting et al Thermostability of class II xylose isomerases  ytị ẳ 1ỵ bk1 k2 À k1 expðÀk1 tÞ À bk1 expðÀk2 tÞ k2 À k1 metals provided It is interesting to note that for the most thermostable enzyme, TNXI, metals were less important for stability than for the other two thermostable enzymes, BLXI and TTXI Unlike BLXI and TTXI, apo- and halo-TNXI melted at similar temperatures (Fig 1) This observation points to the heightened structural rigidity of hyperthermophilic enzymes, even within their functional temperature ranges [48] All XIs are known to be active only in the presence of divalent cations Although there have been reports of XIs active with only one metal site occupied [18], mutating either metal binding site inactivated TNXI In general, activity level varies with substrate, type of metal present, and XI class With glucose as the substrate, class I enzymes are best activated by Mg2+, followed by Co2+ and Mn2+, while class II enzymes are best activated by Co2+, then Mg2+ and Mn2+ In contrast, with xylose as the substrate, class II XIs are best activated by Mn2+ [16] It is interesting that some XIs show maximum activity in the presence of both Mg2+ and Co2+ at ratios of : 1, respectively, or higher [39,43–45,49] The reasons for the differences in the metal preference for specific enzymes and substrates are not clear, but this is most likely related to subtle conformational changes in the active site that vary depending on the specific metal present in each site In addition to maximizing activity, the presence of both Mg2+ and Co2+ also enhanced the thermostability of all the XIs studied when compared with Mg2+ or Co2+ alone While the melting curves of ECXI, BLXI, ð1Þ If there is no active intermediate (E1), b ¼ and the expression reduces to a first order decay Equation (1) was used to fit TNXI inactivation data at different temperatures and at various concentrations and combinations of the three metals (Table 2; Fig 7) Half-lives of the enzymes were calculated by setting y(t) in Eqn (1) equal to 0.5 and solving for t1 ⁄ The inactivation behavior analysis revealed the sensitivity of TNXI kinetic stability to temperature For example, TNXI’s half-life in the presence of Co2+ drops from 59.5 at 96 °C to 14 at 99 °C At 99 °C (i.e close to Tm), TNXI’s estimated t1 ⁄ show that Mn2+ is the most stabilizing metal (t1 ⁄ of 84 min), followed by Co2+ (t1 ⁄ of 14 min) and then Mg2+ (t1 ⁄ of min) Adding 0.5 mm Co2+ to an excess of Mg2+ (5 mm) tripled the t1 ⁄ compared to only Mg2+ Based on inactivation rate constants for the two-step mechanism, the first step proceeded at a much higher rate than the second step (the ratios of rate constants k1 ⁄ k2 were at least 15 for all cases at 99–100 °C) Discussion Despite the structural similarities shared by the class II XIs compared here (all show ‡ 48% sequence identity), there was significant variation in the degree of structural thermostabilization that the different divalent Table Effect of divalent metals cations on TNXI inactivation kinetics  ytị ẳ Aexpk1 tị ỵ Bexpk2 tị or ytị ẳ ỵ bk1 k2 k1 expðÀk1 tÞ À bk1 expðÀk1 tÞ k2 À k1 Metal Conc (mM) T (°C) A k1 (min)1) B k2 (min)1) b t1 ⁄ (min) v2 (· 10)2) r2 Mn2+ Co2+ 99 102 104 106 96 99 102 104 92 96 99 102 87 90 100 100 0.428 0.518 0.639 0.668 0.41 0.43 0.51 0.61 0.534 0.739 0.771 0.946 0.910 1.02 0.42 0.64 0.22 0.40 0.41 0.61 0.08 0.20 0.36 0.49 0.24 0.52 0.57 0.67 0.02 0.06 0.48 0.45 0.58 0.48 0.40 0.34 0.59 0.57 0.49 0.39 0.48 0.29 0.26 0.11 – – 0.58 0.35 0.002 0.005 0.008 0.014 0.003 0.013 0.020 0.038 0.001 0.011 0.027 0.056 – – 0.023 0.017 0.57 0.48 0.39 0.34 0.57 0.53 0.46 0.36 0.48 0.29 0.24 0.10 – – 0.55 0.34 83.2 6.9 4.2 2.3 59.5 14 5.8 2.9 12 2.3 1.9 1.3 30 11.3 7.5 3.0 0.024 0.083 0.224 0.061 0.057 0.058 0.024 0.029 0.080 0.126 0.145 0.384 0.398 0.185 0.014 0.013 0.996 0.983 0.966 0.991 0.992 0.993 0.997 0.997 0.984 0.987 0.986 0.968 0.965 0.996 0.998 0.998 Mg2+ None Mg2+ ⁄ Co2+ Co2+ a a – ⁄ 0.5 0.5 Data from [29] FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS 1459 Thermostability of class II xylose isomerases and TTXI in the presence of two different metals all resembled the single-metal cases, the TNXI curve showed two transitions (Fig 3) TNXI’s unusual melting behavior in the presence of Mg2+ and Co2+ was noted previously [50] Further investigations showed that the relative size of the transitions changed when the pH was increased from 7.0 to 7.9 [51], suggesting that the unusual melting behavior was related to metal binding affinity In fact, TNXI exhibits two melting transitions in the presence of any two of the metals studied (i.e Mn2+, Co2+, and Mg2+) (Fig 3) To confirm that this melting behavior was due to the presence of two different metals in TNXI’s metal binding sites, mutations to site M1 (E232K) and site M2 (D309K) were introduced in TNXI In either case, the enzyme contained at most one metal per active site Both TNXI mutants showed single melting transitions Studies of Streptomyces class I XIs have shown that the metal binding sites (M1 and M2) have different binding affinities [17,52] and that the high and low affinity sites are different for different cations [36]: M1 is the high affinity site for Mg2+, while M2 is the high affinity site for Co2+ and Mn2+ [16] High and low affinity sites were also reported in class II XIs [19], and they are likely to be the same as in class I XIs, since the overall structure and the metal binding residues are conserved across both enzyme classes The TNXI E232K mutant would therefore have higher affinity for Co2+ as only the M2 site is intact, while the D309K mutant would have higher affinity for Mg2+ for similar reasons In the presence of Mg2+ and Co2+, D309K TNXI has a Tm (95.5 °C) slightly lower than that of the apoenzyme, and E323K TNXI has the same Tm as TNXI in the presence of a single metal These results suggest that M2 is the only metal important for TNXI stability, while both metals are needed for activity Cleavage of the Arthrobacter XI C-terminal loop by thermolysin affected neither stability nor activity [53], suggesting that thermal inactivation begins in the TIM barrel Site directed mutagenesis was used in various studies to examine metal binding in XIs (reviewed in [7]) Point mutations that trigger conformational changes in the active site residues destabilize XIs, and mutations that alter the metal binding residues greatly reduce or eliminate activity [21,37,40,54,55] These results suggest that the irreversible thermal unfolding begins with movement of active site residues [56] The metal cofactors are thought to hold the active site in a stable conformation that is lost when the metals are removed [26] While the metals play an essential part in the catalytic mechanism, they are not required for the enzyme to fold properly Indeed, the two metal site mutants constructed in this study behaved exactly as 1460 K L Epting et al TNXI in the different purification steps (in particular, they could be heat-treated in the same conditions as TNXI and remain soluble) They also showed melting transitions as high as the apoenzyme, and their mobility on native PAGE was identical to that of TNXI (data not shown) TNXI showed non-first order kinetic inactivation in the presence of Co2+, Mg2+ or Mn2+ These kinetics suggest a complex, higher order process that can correspond to numerous possible molecular mechanisms [47] TNXI inactivation could be modeled as the sum of two exponentials, as shown in Fig and Table This general mechanism can be interpreted as a sequential inactivation, with one or more catalytically active intermediates Non-first order inactivation has been reported previously for the Thermotoga sp and Streptomyces murinus XIs [29,51,57], although instead of a single model, the inactivation data were divided into two first-order phases – a faster initial phase and a slower later phase However, TNXI showed first order inactivation when covalently immobilized to glass beads [51] or when heated in the absence of metals (apo) These results suggest that the soluble TNXI inactivates through one or more partially active intermediates that are not as active as the native form These intermediates are unable to form when the enzyme is physically attached to a surface Hartley et al [7] proposed that all XIs follow a common heat inactivation pathway involving irreversible conversion to an altered apoenzyme that cannot bind metals, followed by unfolding The pathway can be described as T fi T* fi M fi A, where T is the active tetramer which is converted to the inactive apotetramer (T*) T* then dissociates into monomers (M) that unfold and form aggregates (A), This can be applied to the recombinant TNXI, which exists primarily as a dimer [50], as T « D fi D* fi M fi A Here the tetramer (T) is in an equilibrium with the dimer (D), which is stable until it is irreversibly converted into an apodimer (D*), which can no longer bind metal Inactive dimer formation during heat treatment has been observed for the class I Streptomyces XI [58], for which inactivation was due to a change in the active site region Figure shows that some TNXI remains in its native form throughout the melting transition Presumably, TNXI does not denature until the all the metals are lost This pathway, however, would only describe first order inactivation, as the only active species are the tetramer (T) and dimer (D), which have similar biochemical and biophysical properties for TNXI [50] It is possible that the active intermediate is a form D¢ between D and D*, where only one of the active sites in the dimer is active The pathway could FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS K L Epting et al be represented T « D fi D¢ fi D* fi M fi A, which would explain the non-first order inactivation If each monomer in the dimer inactivated independent of its partner, the relative residual activity of the intermediate (b) would be 0.5 As the subunit interaction of Phe59 is thought to help shield the active site [59], the inactivation of one monomer may shift this important residue, thus decreasing the catalytic activity of the remaining monomer This is supported by the influence of temperature on the TNXI inactivation parameter b (Table 2); at the lower temperatures b is close to 0.5, but as temperature increases b decreases significantly While metals are important in XIs for catalysis, it appears that their influence on structural stability varies While certain divalent cations stabilize some XIs by up to 20 °C, metals played a relatively minor role in the stabilization of the most thermostable (TNXI) XI yet identified The results of this study suggest that subtle modifications in structure at high temperatures that result from dissimilar metals bound to binding sites in TNXI created a structurally stable, but catalytically inept, form of the enzyme Additional efforts with homologous cofactor-requiring enzymes spanning large functional temperature ranges are needed to see if the results observed here are more generally applicable It would also be interesting to see whether forms of TNXI that are structurally stable in the presence of multiple metal cations (second melting transition) could be rendered catalytically viable Experimental procedures Bacterial strains Recombinant TNXI was expressed in E coli BL21(DE3) (Novagen, Madison, WI, USA) carrying plasmid pET22b(+) containing the T neapolitana 5068 xylA gene as an NdeI–HindIII insert (i.e plasmid pTNXI22) [50] E coli HB101 carrying plasmid pCMG11-3 [28] was used to overexpress recombinant TTXI ECXI was expressed in E coli JM105 carrying plasmid pKKX7 [25], while BLXI was expressed in E coli HB101 carrying plasmid pBL2 [26] Enzyme purification All XIs were purified from 1-L cultures grown in LB medium After centrifugation for 10 at 4000 g, the cells were re-suspended in 50 mm Mops (pH 7.0) containing mm MgSO4 plus 0.5 mm CoCl2 (i.e buffer A) The cells were disrupted by two consecutive passes through a French Pressure cell (Thermo Spectronic, Walthum, MA, USA) using a pressure drop of 14 000 p.s.i After centrifugation at 25 000 g, the supernatant was heat-treated for 15 at FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS Thermostability of class II xylose isomerases 85 °C (TNXI), 70 °C (TTXI), 60 °C (BLXI), and 50 °C (ECXI) The precipitated material was separated by centrifugation at 25 000 g for 30 The soluble fraction was loaded on a DEAE–Sepharose Fast-Flow column equilibrated with buffer A The protein was eluted with a linear 0–0.5 m NaCl gradient in buffer A, and the active fractions were analyzed by SDS ⁄ PAGE Partially purified enzymes were loaded onto a Q-Sepharose column and eluded with a linear 0–0.5 m NaCl gradient in buffer A Active fractions were combined and concentrated in a stirred ultrafiltration cell (Amicon, Beverly, MA, USA), dialyzed against buffer A, and stored at °C Protein concentrations were assayed by the method of Bradford [60] using bovine serum albumin as the standard Site directed mutagenesis Point mutations were introduced into the T neapolitana xylA gene using the QuickChangeÔ Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) Residues Glu232 and Asp309 were substituted with Lys to block the metal binding sites The oligonucleotides used for mutagenesis were synthesized by Integrated DNA Technologies (Coralville, IA) Plasmid pTNXI22 was used as the template for mutagenesis Oligonucleotide 5¢-GGACAGTTCCTCATCAAACCAAAA CCGAAAGAACCC-3¢ (mutation site underlined) and its complement were used to construct mutation E232K Oligonucleotide 5¢-CTTCTTCTTGGATGGGACACCAAACAG TTCCCAACAAA-3¢ (mutation site underlined) and its complement were used to construct mutation D309K The double mutant was produced by using the plasmid encoding the E232K mutation as the template and repeating the mutagenesis protocol using the D309K primers Mutations were verified by DNA sequencing performed by the Integrated Biotechnology Laboratories Sequencing and Synthesis Facility (University of Georgia, Athens, GA, USA) The mutant enzymes were expressed in E coli BL21(DE3), and purified following the same procedure as the wild-type enzyme EDTA treatment The purified enzyme was dialyzed overnight at °C against L of 50 mm Mops (pH 7.0) containing 10 mm EDTA It was then dialyzed twice against 50 mm Mops (pH 7.0) containing mm EDTA, and finally dialyzed twice against 50 mm Mops (pH 7.0) The apoenzyme was stored at °C until use Differential scanning calorimetry DSC experiments were performed on a Nano-Cal differential scanning calorimeter (Calorimetry Sciences Corp., Provo, UT, USA) To determine the scan rate, the TNXI enzyme in buffer A was examined using a scan rate of 0.5 1461 Thermostability of class II xylose isomerases and °C min)1 There were no noticeable differences between the results of the scans, therefore a scan rate of °C min)1 was used for all the comparative studies Samples were scanned from 25 °C to 100 °C for ECXI and 25 °C to 125 °C for TTXI and TNXI The reversibility of the thermal transition was checked by reheating the samples after cooling from the first scan The apoenzymes were scanned against 50 mm Mops (pH 7.0) To prepare the single metal-containing enzymes, the apoenzyme was dialyzed at °C overnight against 50 mm Mops (pH 7.0) containing mm metal-chloride The enzyme solution was then dialyzed once against L of 50 mm Mops (pH 7.0) to remove unbound metal and scanned against the corresponding dialysis buffer DSC experiments (with apo- and single metal-containing enzymes) were conducted with 1.3 ± 0.3 mgỈmL)1 (TNXI), 1.2 ± 0.6 mgỈmL)1 (TTXI), and 2.0 ± 0.7 mgỈmL)1 (ECXI) For mixed-metal DSC experiments, the apoenzyme was dialyzed against 50 mm Mops (pH 7.0) containing mm MgSO4 plus either 0.5 mm CoCl2 or mm MnCl2 and scanned against the dialysis buffer Enzyme concentrations were 0.9 mgỈmL)1 (TNXI), 1.7 mgỈmL)1 (TTXI), and 3.0 mgỈmL)1 (ECXI) Higher concentrations were used for ECXI because ECXI gave a weaker signal during its thermal transition Enzyme assays Enzyme activity was assayed routinely with glucose as the substrate TNXI (10–20 lg) was incubated in 200 lL of 50 mm Mops (pH 7.0 at room temperature) containing mm CoCl2 and 1.0 m glucose at 80 °C for 10 The reaction was stopped by transferring the tube to an ice bath The amount of fructose produced was determined by the resorcinol–ferric ammonium sulfate–hydrochloric acid method [61] Enzyme kinetic inactivation To determine the effect of specific metals on TNXI kinetic stability, the apoenzyme (100–200 lgỈmL)1 final concentration) was preequilibrated with 0.5 mm CoCl2, 0.5 mm MnCl2, or mm MgCl2 in 50 mm Mops (pH 7.0) for 30 at 30 °C (preincubation conditions that are known to be sufficient for the metal to reach equilibrium between the buffer and enzyme metal-binding sites [19]) One hundred lL aliquots of the enzyme solution were then incubated in 0.1 mL MultiplyÒ-Safecup screw-cap microtubes (Sarstedt, Newton, NC, USA) at various temperatures in an oil bath for different periods of time Inactivation was stopped by transferring tubes to a room-temperature water bath Residual activity was determined using the assay described above Non-linear curve fitting of the inactivation data was performed using the v2 minimization procedure of the origin software (Microcal Software, Inc., Northampton, MA, USA) 1462 K L Epting et al Acknowledgements This work was supported in part through grants from the NSF to RMK (Bes-0115734 and Bes-0317886), and to CV ⁄ JGZ (Bes-0115754) References Vieille C & Zeikus J 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utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 Schenk M & Bisswanger H (1998) A microplate assay for d-xylose ⁄ d-glucose isomerase Enzyme Microb Technol 22, 721–723 FEBS Journal 272 (2005) 1454–1464 ª 2005 FEBS ... enzyme’s inactivation kinetics, the inactivation course of TNXI in the presence of various metal concentrations and metal combinations was determined Figure shows the inactivation courses of apo-TNXI... courses of apo-TNXI and of TNXI in the presence of mm concentrations of each of the three divalent cations (Mg2+, Mn2+, or Co2+) With the exception of apo-TNXI, whose inactivation was first-order,... (kcal/mole·K) Thermostability of class II xylose isomerases TTXI ECXI TNXI TNXI* Fig Effect of metals on the Tm values of class II XIs DT is the difference between Tm (enzyme in the presence of lM single