Báo cáo khoa học: Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium pdf

Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature Radka Chaloupkova 1,2 , Zbynek Prokop 1,2 , Yukari Sato 3 , Yuji Nagata 3 and Jiri Damborsky 1,2 1 Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 2 International Clinical Research Center, St Anne’s University Hospital Brno, Czech Republic 3 Graduate School of Life Sciences, Tohoku University, Sendai, Japan Keywords activity; enantioselectivity; haloalkane dehalogenase; oligomerization; pH; structure; thermostability Correspondence J. Damborsky, Loschmidt Laboratories, Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5 ⁄ A13, 625 00 Brno, Czech Republic Fax: +420549496302 Tel: +420549493467 E-mail: jiri@chemi.muni.cz (Received 6 February 2011, revised 15 May 2011, accepted 31 May 2011) doi:10.1111/j.1742-4658.2011.08203.x The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3–10.3 and 6.2–10.1, respectively. Oligomeri- zation of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solu- tion at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting tempera- ture (T m = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7–10.4. While pH did not have any effect on enantiodiscri- minination of DbjA, temperature significantly altered DbjA enantioselectiv- ity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropi- onate and ethyl 2-bromobutyrate, and differential activation parameters D RÀS DH z and D RÀS DS z were determined. The thermodynamic analysis revealed that the resolution of b-bromoalkanes was driven by both enthal- pic and entropic terms, while the resolution of a-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stabil- ity of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC 3.8.1.5 haloalkane dehalogenase. Abbreviations CD, circular dichroism; MRE, mean residue ellipticity. 2728 FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS Introduction Haloalkane dehalogenases (EC 3.8.1.5) make up an important class of enzymes which are able to cleave car- bon–halogen bonds in a broad range of halogenated ali- phatic compounds. The hydrolytic dehalogenation catalysed by these enzymes proceeds by nucleophilic substitution of a halogen atom with a hydroxyl group forming corresponding alcohols [1]. Haloalkanes, halo- alcohols and alcohols are valuable building blocks in organic and pharmaceutical synthesis [2–4], making haloalkane dehalogenases potentially applicable in bio- catalysis. We have recently shown that newly isolated haloalkane dehalogenase DbjA from Bradyrhizobium ja- ponicum USDA110 [5] possesses new substrate specific- ity with high catalytic activity towards b-methylated haloalkanes and sufficient enantioselectivity for indus- trial scale synthesis of optically pure compounds [6]. Interestingly, the haloalkane dehalogenase DbjA (a) can kinetically discriminate between enantiomers of two dis- tinct groups of substrates, a-bromoesters and b-bro- moalkanes; (b) has enantioselectivity based on distinct molecular interactions, which can be modified sepa- rately by engineering of a surface loop; and (c) can adopt an inverse temperature dependence of enantiose- lectivity for b-bromoalkanes, but not a-bromoesters, by mutating this surface loop and a flanking residue [7]. Use of enzymes in biocatalytic preparation of opti- cally pure substances has been rapidly expanding in recent years [8]. The efficient utilization of enzymes in industrial processes requires that a number of criteria are fulfilled, e.g. high activity, stability under process conditions, appropriate substrate specificity and enanti- oselectivity [9–11]. The manipulation of the physical environment is an attractive way to provide additional control of enzyme stereochemistry and catalytic func- tionality alongside other methods, such as protein engineering and directed evolution [12–14]. Under- standing the effect of physical parameters on the struc- ture and activity of an enzyme is important for optimization of the operational conditions of a biocat- alytic process, while knowledge of the structure–func- tion relationships provides an essential theoretical framework for modification of a biocatalyst by rational protein design [15]. In this work we have systematically examined the effects of pH and temperature on the stability, oligo- merization state and functionality of the DbjA enzyme using CD spectroscopy, size exclusion chromatogra- phy, activity and enantioselectivity assays. Thermody- namic analysis has been used to address the origin of enantiomeric discrimination by determining differential activation enthalpy and entropy for the enzymatic reaction with racemic substrates 2-bromobutane, 2-bromopentane, ethyl 2-bromopropionate and methyl 2-bromobutyrate. Results and Discussion Conformational changes CD spectroscopy was used for investigation of the sec- ondary and tertiary structure of the DbjA enzyme at pH conditions ranging from 1.7 to 11.5 in the far UV and near UV spectral regions, respectively. The far UV CD spectrum of native enzyme, measured in 50 m M potassium phosphate buffer (pH 7.5 at 4 °C), exhibited two negative features at 208 and 222 nm characteristic of a-helical content (Fig. 1A, red bold curve). Similar spectral features were found throughout the pH range 5.3–10.3, suggesting that enzyme secondary structure remained preserved under these conditions. Calculated Fig. 1. Far UV (A) and near UV (B) CD spectra of DbjA as a func- tion of pH. The spectra shown represent the average of 10 consec- utive scans. R. Chaloupkova et al. Stereochemistry and conformational stability of DbjA FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS 2729 a-helical content as a function of pH using the method of Chen et al. [16], which is based on far UV CD data at 222 nm, is presented in Fig. 2. Predicted a-helical content at pH 5.3–10.3 was about 30.5%. The second- ary structure of DbjA remains intact within five pH units. At lower pH levels (pH < 5.0), the enzyme visu- ally aggregates with simultaneous loss of UV signal. On the other hand, at pH 11.0–11.4, the enzyme stays in solution showing approximately a 42% loss in a-helical content in comparison with its native state. A strong negative band at 204 nm and a weak band at 220 nm suggest that DbjA enzyme conformation starts to be disordered at these extremely alkaline conditions. The near UV CD spectrum of the native state of the enzyme reveals three negative ellipticity peaks at 259, 265 and 285 nm and a positive peak at 292 nm (Fig. 1B, red bold curve). The ellipticity values at these wavelengths remain preserved within the pH range 6.2–10.1. In acidic conditions, pH < 6.2, the CD intensity at 285 and 292 nm slightly increases as a result of the decreasing pH. The positive ellipticity at 292 nm can be attributed to a tryptophan environment, since this region corresponds to the absorption band for this residue [17]. The intensity changes observed at 292 nm might be related to a change in the tryptophan environment as a result of the loss of some tertiary interactions. This indicates that the enzyme starts to lose its tertiary interactions without any secondary structure loss before complete aggregation. In alkaline conditions, pH > 10.7, the protein loses most of its tertiary structure. A considerable increase in the ellip- ticity at pH ‡ 10.7 is observed at 259 nm. This could be caused by sudden exposure of phenylalanine resi- dues in the extreme alkaline pH region. Comparison of both near UV and far UV CD spectra determined at various pH conditions revealed similar pH regions at which the enzyme is structurally stable. Changes in the structure could be attributed to a change of ionization state of the enzyme at pH condi- tions close to its isoelectric point (pI). The predicted pI of DbjA is 5.89. Although many proteins demonstrate a state of minimal solubility at their pI conditions, DbjA remains soluble with a preserved secondary structure. When pH is decreased below 5.3, the enzyme suddenly passes from a nearly native state which is sol- uble to a completely aggregated state. On the other hand, alkalic denaturation of DbjA is accompanied by significant modification of both secondary and tertiary structure. At pH conditions 10.3–11.5, the enzyme occurs in disordered conformation and remains soluble. Temperature dependence of conformational stability was evaluated by performing a thermal unfolding experiment at different pH conditions. Dependence of the melting temperature on pH was monitored by CD spectroscopy at 222 nm (Fig. 2). All thermal transi- tions obtained were irreversible, possibly because of the aggregation phenomena in the denatured state where visible aggregates were observed after heating of the enzyme sample up to 80 °C. The pH dependence exhibits a bell-shaped curve with the highest T m (49.1 ± 0.2 °C) at pH 6.5. A decrease in DbjA ther- mostability at pH below 6.5 possibly corresponds to the loss of tertiary interactions, as indicated by CD spectra determined in the near UV spectral region. On the other hand, the decrease in the enzyme thermosta- bility at a pH above 6.5 could be attributed to the changes in the protonation state of the enzyme, since no changes in enzyme structure were observed in this pH region. Generally, two major factors are known to determine optimal pH for protein stability: amino acid composition and tertiary structure [18]. In addition, we suggest that quaternary structure can also influence thermal stability of proteins. Oligomerization Analytical gel filtration was used to quantitatively assess the effect of pH on the oligomerization state of DbjA. Monomer, dimer, tetramer and high molecular weight clusters were distinguished by enzyme elution Fig. 2. pH-dependent dissociation, deactivation and denaturation of DbjA: , melting temperature evaluated from measured changes in ellipticity at 222 nm with increasing temperature; m, relative activity (in %) representing the portion of the maximal detected specific activity (lmolÆs )1 Æmg )1 ) at a particular pH; , near UV CD at 259 nm; h, a-helical content calculated by the method of Chen et al. [16] based on far UV CD at 222 nm. Stereochemistry and conformational stability of DbjA R. Chaloupkova et al. 2730 FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS volume at different pH conditions (Fig. 3). While the pure monomer of DbjA is found under the lowest tested pH conditions (pH 5.9), the dimeric form is a dominant species at pH conditions equal to or higher than 6.1. As pH increases, both dimeric and tetrameric forms are present in solution. Abundance of the tetrameric form gradually increases until it prevails at pH 9.6 (Fig. S1). A high molecular weight cluster appears in solution as another oligomeric form of DbjA at pH conditions higher than 9.6. The presence of this cluster most prob- ably corresponds to change in the conformation of the enzyme detected by CD spectroscopy. In these alkaline conditions, the DbjA enzyme occurs in a predominantly unordered conformation which leads to association of the enzyme to a high molecular weight cluster. Associa- tion of oligomeric proteins at extreme conditions proba- bly represents protection against aggregation. These results demonstrate that oligomerization of DbjA in solution strongly depends on the pH of the surrounding environment. One of the major driving forces for oligomerization comes from shape comple- mentarity between the associating molecules, brought about by a combination of hydrophobic and polar interactions [19]. As determined by gel filtration, the enzyme is monomeric at conditions close to its pI (5.89). This suggests that the monomer is predomi- nantly favoured at a pH where the net charge of the enzyme is equal to zero. Under these conditions, all oligomer-forming residues contribute to the overall enzyme electronegativity via intramolecular interac- tions and for that reason they do not contribute to the formation of oligomers. As pH increases above pI, the enzyme starts to be more and more negatively charged and its oligomeric form is favoured. The enzyme occurs in different ratios of dimeric and tetrameric forms in the pH range 6.5–9.6. Under these conditions, charged interface residues may establish intermolecular interactions leading to the formation of a DbjA oligo- mer. Crystallographic analysis [7] of the DbjA struc- ture revealed that subunits of the dimer interact predominantly in two regions: the C-terminal part of the last helix (R292–P306) and the b-strand 8 region (R269–L275). As was evident from measured T m at different pH conditions, oligomeric states of DbjA obviously influ- ence its thermal stability at different pH conditions. The highest thermostability of the enzyme was detected at pH 6.5, when the dimeric form predominates in solution. With increasing occurrence of the tetrameric form in solution, thermal stability of the enzyme decreases. DbjA thermostability also slightly decreases at pH 5.7–5.9, when the enzyme is monomeric. This suggests that different forms of DbjA have different thermostabilities: T m (dimer) > T m (monomer) > T m (tetramer). The stability of a high molecular weight cluster present in solution above pH 9.6 is not dis- cussed because its occurrence is accompanied by con- formational changes which naturally lead to destabilization of the protein structure. Fig. 3. Gel filtration chromatograms of solutions with DbjA at dif- ferent pH conditions. The peaks marked I, II, III and IV represent monomer, dimer, tetramer and a high molecular weight cluster, respectively. Molecular weight (MW) standards (Fig. S2) included ribonuclease A (13.7 kDa, line 1), ovalbumin monomer (43.0 kDa, line 2), albumin monomer (67.0 kDa, line 3), ovalbumin dimer (86.0 kDa, line 4) and albumin dimer (134.0 kDa, line 5). Blue Dex- tran (line 6) was used for determination of the dead volume of the gel filtration column. R. Chaloupkova et al. Stereochemistry and conformational stability of DbjA FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS 2731 pH profile Measurement of DbjA activity was performed to explore whether catalytic function directly relates to conformational stability at various pH values. Experi- ments were done under different pH conditions and saturated concentrations of substrate 1-iodohexane for which DbjA exhibited the highest catalytic efficiency [5]. The activity profile of this enzyme shows a maxi- mum at pH 9.7 (Fig. 2). However, the enzyme retains at least 90% of its maximum activity at pH conditions ranging from 7.7 to 10.4. DbjA thus possesses the broadest pH optimum compared with other biochemi- cally characterized haloalkane dehalogenases (Fig. 4). This phenomenon is most likely related to the fact that DbjA occurs as oligomer. The melting temperatures of DbjA detected at optimal pH represent only 79.1% of maximal T m . For this reason, the pH interval at which the enzyme possesses the highest activity and the high- est thermostability simultaneously is narrowed to between pH 7.4 and 8.7 (Fig. 2). DbjA activity decreases below pH 7.0 and above pH 10.4 with no activity detected below pH 5.0 and above pH 11.0. These results correlate well with the conformational stability as a function of pH observed by CD spectros- copy. The loss of enzymatic activity at highly alkalic conditions is caused by change from native to predom- inantly disordered conformation. The drop in activity below pH 7.0 is not induced by the structural changes but by change in the protonation state of catalytic amino acids. Catalytic residues of DbjA comprise five key resi- dues forming the so-called catalytic pentad [1]. The catalytic pentad of DbjA consists of three residues involved in the catalytic reaction, Asp103, Glu127 and His280, and two H-bond donating residues, Asn38 and Trp104, involved in stabilization of a halogen group of the substrate. With respect to particular dissociation constants of catalytic residues, pK Asp a = 3.90 (b-COOH), pK Glu a = 4.07 (c-COOH), pK His a = 6.04 (imidazol) [20], it is evident that the residue affecting the enzyme activity below pH 7.0 is His280. At pH 6.1, the enzyme retains 50% of its maximal activity which nicely corresponds to pK His a . Under these condi- tions, 50% of histidine is protonated and thus non- reactive and 50% is still reactive. The imidazol ring of His becomes protonated and the enzyme loses its activ- ity when the pH decreases further. Knowledge of the pH interval at which the enzyme retains its structure but loses most of its activity due to protonation of cat- alytic histidine is interesting for further detailed deter- mination of its catalytic mechanism. An alkyl–enzyme intermediate can be captured by protein crystallogra- phy at these pH conditions as has been previously described for the haloalkane dehalogenase DhlA [21]. The effect of pH on enantioselectivity The dependence of DbjA enantioselectivity on pH was tested in a reaction with 2-bromopentane. Although the effect of pH on enzyme enantioselectivity has already been described for both charged [22] and uncharged [23] substrates, in the case of DbjA no sig- nificant change in enantioselectivity was observed at pH values ranging from 6.7 to 10.1 (data not shown). Results indicated that ionization of the alkyl–enzyme intermediate is the same for both enantiomers at all tested pH values and corresponds with the theoretical Enzymes pH 678910 DhlA a DhaA b LinB c DhmA d DmbA e DmbB e DmbC f DrbA f DbjA g Fig. 4. Comparison of the pH profiles of biochemically characterized haloalkane dehalogenases. Enzyme activity was quantified as the spe- cific enzyme activity in units of lmolÆs )1 Æmg )1 under conditions corresponding to initial velocity measurements. Black boxes represent maxi- mal dehalogenating activity. Grey boxes represent retained dehalogenating activity at the level of at least 90% of the maximal enzymatic activity. a DhlA from Xanthobacter autotrophicus GJ10 [24]; b DhaA from Rhodococcus sp. [25]; c LinB from Sphingobium japonicum UT26 [39]; d DhmA from Mycobacterium avium N85 [26]; e DmbA and DmbB from Mycobacterium bovis 5033 ⁄ 66 [27]; f DmbC from Mycobacte- rium bovis 5033 ⁄ 66 and DrbA from Rhodopirellula baltica SH1 [28]; g DbjA from Bradyrhizobium japonicum USDA110, this study. Stereochemistry and conformational stability of DbjA R. Chaloupkova et al. 2732 FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS rule that pH dependence of stereoselectivity can only be observed around the pK values of groups in the active site whose ionization controls the enzyme activ- ity [23]. Ionization of the catalytic His of DbjA could be reflected at tested pH conditions, although this effect on enantioselectivity was not observed. The pK a values of other catalytic amino acids of DbjA, i.e. nucleophile Asp and catalytic acid Glu, are lower than the pH conditions at which the enzyme aggregates. Temperature profile Measurement of enzymatic activity at different temper- atures was carried out to study the effect of tempera- ture on the rate of the dehalogenation reaction. The enzyme exhibited the highest activity at 50 °C, although above this temperature it became rapidly inactivated. This observation is in good agreement with similar experiments previously described for other haloalkane dehalogenases possessing the highest activ- ity at temperatures ranging from 35 to 50 °C [24–28]. Thermodynamic analysis of enantioselectivity The temperature dependence of DbjA enantioselectivi- ty was studied to determine differential activation parameters, enthalpy (D RÀS DH z ) and entropy (D RÀS DS z ), contributing to the kinetic resolution of selected b-bromoalkanes (2-bromobutane and 2-brom- opentane) and a-bromoesters (methyl 2-bromopropio- nate and ethyl 2-bromobutyrate). The temperature dependence of DbjA enantioselectivity was measured in the temperature range from 20 to 50 °C. The E val- ues and the thermodynamic components of enantiose- lectivity determined based on the linear relation of ln E and T )1 are summarized in Table 1. Although the studied temperature interval was relatively small, highly significant changes in DbjA enantioselectivity were observed. Variation of the reaction temperature from 20 to 50 °C caused a decrease in E value of DbjA from 174 to 13 in the reaction with 2-bromopentane, from 474 to 197 with ethyl-2-bromopropionate and from 225 to 83 with methyl 2-bromobutyrate. Since enzyme enantioselectivity is defined as the ratio of the specificity constants for (R)) and (S)) enantiomers, the E value does not depend on the degree of conver- sion or variation of the reaction mechanism of individ- ual enantiomers with temperature. It should be noted that the enthalpic and the entropic components of dif- ferential activation free energy (D RÀS DG z ) both con- tribute to the overall success of the kinetic resolution of enantiomers [29,30]. All substrates have a racemic temperature significantly above the experimental tem- perature indicating that the entropic component coun- teracts the enthalpic component of enantiomeric discrimination. The linearity between ln E and T )1 observed from 20 to 50 °C suggested that a single tran- sition state structure is held in this temperature range for all tested substrates. Enantiomeric discrimination of 2-bromobutane was not observed at any tested temperature (Fig. 5). This Table 1. Thermodynamic components for the dehalogenation of selected halogenated compounds catalyzed by DbjA. Errors were calculated from the standard errors of the linear regression ln E versus T )1 . T r is the racemic temperature at which no stereochemical discrimination of the enzyme between the (R)) and (S)) enantiomers occurs, E = 1 and D RÀS DG z = 0. It is defined by the ratio of the differential activation enthalpy and entropy, T r ¼ D RÀS DH z =D RÀS DS z , and is constant for a particular racemic substrate converted by a particular enzyme [29,31]. No enantioselectivity was observed for 2-bromobutane. Substrate E, 298 K D RÀS DH z (kJÆmol )1 ) D RÀS DS z (JÆmol )1 ÆK )1 ) T D RÀS DS z , 298 K (kJÆmol )1 ) D RÀS DG z , 298 K (kJÆmol )1 ) T r (°C) 2-Bromobutane 1 – – – – – 2-Bromopentane 132 )69.5 ± 2.6 )193.8 ± 8.4 )57.8 ± 2.5 )11.7 86 Ethyl 2-bromopropionate 392 )24.1 ± 1.8 )31.2 ± 5.9 )9.3 ± 1.8 )14.8 497 Methyl 2-bromobutyrate 209 )25.8 ± 2.2 )42.3 ± 7.2 )12.6 ± 2.1 )13.2 337 Fig. 5. The temperature dependence of enantiomeric ratios determined for dehalogenation of selected b-bromoalkanes (2-bromo- butane, 2-bromopentane) and a-brominated esters (ethyl 2-bromopro- pionate, methyl 2-bromobutyrate) catalyzed by DbjA. R. Chaloupkova et al. Stereochemistry and conformational stability of DbjA FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS 2733 result excludes the possibility that the absence of DbjA enantioselectivity towards 2-bromobutane is due to the fact that the initial E value was determined at a tem- perature (20 °C) close to the racemic temperature for this particular enzymatic resolution. If this were the case, the enantioselectivity of DbjA could be increased with increasing reaction temperature, changing also the enantio-preference of the enzyme [31]. However, our measurements confirm that the absence of 2-bromobu- tane discrimination is the effect of zero D RÀS DG z at all tested temperatures. (R)) and (S)) enantiomers of this simple chiral molecule are probably too similar to each other to be kinetically recognized by the enzyme. Surprisingly, adding a single carbon atom to a substrate molecule provided enough structural dif- ference for high enantiomeric discrimination as was seen in the case of 2-bromopentane (Fig. 5). This finding indicates the importance of the length of the b-substituted bromo-n-alkanes for their kinetic resolu- tion. The temperature dependence of DbjA enantiose- lectivity for 2-bromopentane revealed that both thermodynamic parameters, D RÀS DH z and D RÀS DS z , where the entropic term represents 83% of the enthal- pic term, are important for enantiodiscrimination (Table 1). The high contribution of entropy indicates the importance of solvation, conformational degrees of freedom of the protein, or restriction of substrate motion in the transition state of the reaction. b-bro- moalkanes display high flexibility within the enzyme active site which is related to the significant influence of D RÀS DS z for their kinetic resolution by the DbjA enzyme. This implies that enantiomeric recognition of b-bromoalkanes by DbjA is mediated by the differen- tial conformational freedom of enantiomers upon binding and ⁄ or a displacement of a different number of active site water molecules by the (R)) and (S)) enantiomer [32,33]. The temperature dependence of DbjA enantioselec- tivity with ethyl 2-bromopropionate and methyl 2-bro- mobutyrate revealed that differential activation enthalpy represents a major contribution to their discrimination (Table 1). The high contribution of enthalpy is related to differences in the complementar- ity of each enantiomer in the transition state compris- ing steric and electrostatic interactions between the enzyme active site, its substrate and the solvent. a-bromoesters obviously possess limited flexibility inside the active site cavity due to their ability to form an additional hydrogen bond of a carboxylic oxygen with halide stabilizing residues. This implies that DbjA enantioselectivity towards a-bromoesters is due to different interactions of individual enantiomers with the residues of the enzyme active site in the Michaelis complex and ⁄ or the transition state of the dehalogen- ation reaction [34]. The thermodynamic analysis showed that DbjA enantioselectivity towards b-bromoalkanes and a-bromoesters is differently influenced by individual thermodynamic contributions, differential activation enthalpy and entropy. The resolution of b-bromoalk- anes was found to be driven by both enthalpic and entropic terms, while the resolution of a-bromoesters was driven mainly by an enthalpic term. These results correspond well with the proposal that enantioselectivi- ty of DbjA with b-bromoalkanes and a-bromoesters is based on two distinct molecular interactions [7]. Conclusions Here we show that DbjA possesses unusually high structural and functional stability towards a broad range of pH conditions. Oligomerization of DbjA is strongly pH dependent. Monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions and each oligomeric state demonstrated different stability. The highest thermostability occurred at pH conditions when the enzyme occurs in its dimeric form. Tempera- ture significantly alters enantioselectivity, but an effect of pH on DbjA enantiodicrimination was not observed. Lowering the temperature results in considerable enhancement of enantioselectivity. The results from thermodynamic analysis are in good agreement with the proposal that enantiomeric discrimination of b-bromi- nated alkanes and a-brominated esters by DbjA is controlled by distinct molecular interactions [7]. These results indicate unique properties of DbjA compared with other known and characterized members of haloal- kane dehalogenases. Catalytic activity and structural stability in a broad range of pH conditions combined with high enantioselectivity with selected substrates make DbjA a very versatile biocatalyst. Experimental procedures Enzyme preparation The His-tagged DbjA was overexpressed in Escherichia coli BL21 using a previously described method [5] and purified using the HighTrap Chelating HP 5-mL column charged with Ni 2+ ions (GE Healthcare, Uppsala, Sweden). The enzyme was bound to the resin in equilibrating buffer (20 m M potassium phosphate buffer, pH 7.5, containing 0.5 M sodium chloride and 10 mM imidazole). Unbound and weakly bound proteins were washed out with the buf- fer containing 10 m M imidazole. The target enzyme was Stereochemistry and conformational stability of DbjA R. Chaloupkova et al. 2734 FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS eluted by a buffer containing 500 mM imidazole. The active fractions were pooled and dialysed against a 50 m M potas- sium phosphate buffer (pH 7.5). The enzyme was kept at 4 °C during the purification procedure and stored in 50 m M phosphate buffer at 4 °C until use. CD spectroscopy CD spectra were recorded at room temperature (22 °C) using a Jasco J-810 spectrometer (Jasco, Tokyo, Japan). All the spectra were obtained at an interval of 0.1 nm with a scanning speed of 100 nmÆmin )1 , 1 s response time and 2 nm bandwidth. Cuvettes of 0.1 and 1 cm path length were used in the far and near UV regions, respectively. The protein concentrations for the far UV and the near UV spectra acquisition were 0.23 mgÆmL )1 and 1.15 mgÆ mL )1 , respectively. Each spectrum shown is the average of 10 indi- vidual scans and has been corrected for baseline noise. CD spectra were expressed in millidegrees. The a-helical content of the enzyme was calculated from the mean residue ellip- ticity (MRE) value at 222 nm using the following equation as described by Chen et al. [16]: a -helix % ¼ MRE 222 À2340 30300  100 ð1Þ Thermal denaturation Thermal unfolding of DbjA was followed at different pH conditions by monitoring the ellipticity at 222 nm over the temperature range 20–80 °C, with a resolution 0.2 °C, at a heating rate 0.5 °CÆ min )1 . Recorded thermal denaturation curves were roughly normalized to represent signal changes between $ 1 and 0, and fitted to sigmoidal curves using software ORIGIN 6.1 (OriginLab, Northampton, MA, USA). The melting temperatures (T m ) were evaluated as the mid- point of the normalized thermal transition. Prediction of the isoelectric point The theoretical isoelectric point (pI) of DbjA was predicted based on the amino acid sequence by using EXPASY SERVER [35–37]. Effect of pH DbjA activity and enantioselectivity were measured at dif- ferent pH conditions. Britton–Robinson buffer solutions were used to cover the pH range 1.7–11.5. The solutions were prepared by mixing 0.04 M phosphoric, boric and ace- tic acid with the appropriate volume of sodium hydroxide (0.2 M) and sodium perchlorate monohydrate to get a con- stant ionic strength of 0.15 M. The assays were performed with 1-iodohexane as the substrate for activity measurement at 37 °C or 2-bromopentane as the substrate for enantiose- lectivity measurement at 25 °C. Effect of temperature The effect of temperature on DbjA activity and enantiose- lectivity was determined by performing activity and enanti- oselectivity assays at different temperatures. The activity measurements were evaluated at temperatures ranging from 20 to 60 °C and the enantioselectivity of the DbjA enzyme was monitored in the temperature range 20–50 °C, both in 50 m M glycin buffer at pH 8.6. Activity measurements were performed with 1-iodohexane, and enantioselectivity mea- surements with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate. Gel filtration chromatography The molecular mass of DbjA enzyme at different pH condi- tions was analysed using the FPLC system A ¨ KTA (GE Healthcare) equipped with UV 280 detection (GE Healthcare, Uppsala, Sweden) and SuperdexÔ 200 10 ⁄ 300 GL column (GE Healthcare, Uppsala, Sweden). A total volume of 100 lL of each protein sample was applied to the column and separated at a constant flow rate of 0.5 mLÆmin )1 . Britton–Robinson buffer with an appropriate pH value was used as the mobile phase. The molecular weight standards from the Gel Filtration Calibration Kit (GE Healthcare, Uppsala, Sweden) included ribonuclease A (13.7 kDa), oval- bumin monomer (43.0 kDa), albumin monomer (67.0 kDa), ovalbumin dimer (86.0 kDa) and albumin dimer (134.0 kDa). The dead volume of the SuperdexÔ 200 10 ⁄ 300 GL column was determined using the Blue Dextran of the calibration kit. All protein standards as well as enzyme samples were trans- ferred into the Britton–Robinson buffer by using a 5-mL HighTrap Desalting Sephadex G-25 Superfine column (GE Healthcare, Uppsala, Sweden). Activity assay DbjA activity was assayed by the colorimetric method developed by Iwasaki et al. [38]. The halide ions released were analysed after a reaction with mercuric thiocyanate and ferric ammonium sulfate spectrophotometrically at 460 nm using the Sunrise microplate reader (Tecan, Gro ¨ dig ⁄ Salzburg, Austria). The dehalogenation reaction was performed in 25-mL Reacti flasks closed by Miniert valves at various temperatures. The reaction mixture was composed of 15 mL of buffer and 2 lL of substrate 1-iod- ohexane. The reaction was initiated by the addition of enzyme in a final concentration of 0.15 l M. The reaction was monitored by withdrawing 1 mL samples at 10, 20, 30, 40, 50 and 60 min from the reaction mixture. The reac- tion mixture samples were immediately mixed with 0.1 mL 35% nitric acid to terminate the reaction. Dehalogenation activity was quantified as a rate of product formation in time. Each activity was measured in three to five indepen- dent replicates and represented as mean values of relative R. Chaloupkova et al. Stereochemistry and conformational stability of DbjA FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS 2735 activity with plotted standard errors. Relative activities represented a percentage of maximal specific activity detected. Enantioselectivity assay Enantioselectivity was analysed in 25-mL Reacti flasks closed by Miniert valves containing 20 mL of glycin buffer (100 m M, pH 8.6). Chiral substrates were added to a final concentration of 0.5–3.0 m M with regard to enzyme affinity. The enzymatic reaction was initiated by the addition of appropriate amounts of the DbjA enzyme depending on enzyme activity (final concentration 0.2–2.0 l M). The reac- tion was monitored by periodical withdrawing of 0.5 mL sample aliquots from the reaction mixture. The reaction was stopped by mixing the sample with 1 mL of diethyl ether containing 1,2-dichloroethane as an internal standard. After extraction, diethyl ether was anhydrated on a glass column with sodium sulphate. The samples were automati- cally analysed by using Hewlett-Packard 6890 gas chro- matograph (Agilent, Santa Clara, USA) equipped with a flame ionization detector and chiral capillary column Chi- raldex B-TA and Chiraldex G-TA (Alltech, Deerfield, USA). Michaelis–Menten parameters were derived by fitting the progress curves obtained from kinetic resolution experi- ments into a competitive kinetic pattern by numerical integration using the software MICROMATH SCIENTIST (ChemSW, Fairfield, USA). Enantioselectivity was deter- mined as the enantiomeric ratio (E) defined by E ¼ k R cat =K R m k S cat =K S m ð2Þ where k cat and K m represent the Michaelis–Menten parame- ters of the two enantiomers. Thermodynamic analysis The difference in activation enthalpy and entropy between enantiomers was determined by studying the variation of the enzyme enantiomeric ratio with temperature: lnE ¼À D RÀS DH z R Á 1 T þ D RÀS DS z R ð3Þ The enantiomeric ratio (or rather lnE) varied with recipro- cal temperature to an extent determined by the enthalpic term (the slope of Eqn 3, D RÀS DH z ⁄ R), at a level deter- mined by the entropic term (the intercept of Eqn 3, D RÀS DS z ⁄ R). 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Stereochemistry and conformational stability of DbjA FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS 2737 [...]...Stereochemistry and conformational stability of DbjA R Chaloupkova et al Supporting information The following supplementary material is available: Fig S1 Distribution of various forms of DbjA in solution at different pH conditions Fig S2 Gel filtration chromatogram of ribonuclease A (13.7 kDa, line 1), ovalbumin monomer (43.0 kDa, line 2), albumin monomer (67.0 kDa, line 3), ovalbumin dimer (86.0 kDa, line 4) and. .. 5) used as molecular weight standards Blue Dextran (line 6) was used for determination of dead volume of the gel filtration column 2738 This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be reorganized for online... provides supporting information supplied by the authors Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 2728–2738 ª 2011 The Authors Journal compilation ª 2011 FEBS . Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature Radka Chaloupkova 1,2 ,. effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes. (A) and near UV (B) CD spectra of DbjA as a func- tion of pH. The spectra shown represent the average of 10 consec- utive scans. R. Chaloupkova et al. Stereochemistry and conformational stability