Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase

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Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase
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  Bivalent cations and amino-acid composition contribute to thethermostability of  Bacillus licheniformis   xylose isomerase Claire Vieille 1 , Kevin L. Epting 2 , Robert M. Kelly 2 and J. Gregory Zeikus 1 1  Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA;  2  Department of ChemicalEngineering, North Carolina State University, Raleigh, NC, USA Comparative analysis of genome sequence data frommesophilic and hyperthermophilic micro-organisms hasrevealed a strong bias against specific thermolabile amino-acid residues (i.e. N and Q) in hyperthermophilic proteins.The N  þ  Q content of class II xylose isomerases (XIs)from mesophiles, moderate thermophiles, and hyperther-mophiles was examined. It was found to correlateinversely with the growth temperature of the sourceorganism in all cases examined, except for the previouslyuncharacterized XI from  Bacillus licheniformis  DSM13(BLXI), which had an N  þ  Q content comparable to thatof homologs from much more thermophilic sources. Todetermine whether BLXI behaves as a thermostableenzyme, it was expressed in  Escherichia coli,  and thethermostability and activity properties of the recombinantenzyme were studied. Indeed, it was optimally active at70–72  8 C, which is significantly higher than the optimalgrowth temperature (37  8 C) of   B. licheniformis.  Thekinetic properties of BLXI, determined at 60 8 C withglucose and xylose as substrates, were comparable tothose of other class II XIs. The stability of BLXI wasdependent on the metallic cation present in its twometal-binding sites. The enzyme thermostabilityincreased in the order apoenzyme  ,  Mg 2 þ –enzyme  , Co 2 þ –enzyme  <  Mn 2 þ –enzyme, with melting tempera-tures of 50.3 8 C, 53.3  8 C, 73.4  8 C, and 73.6  8 C. BLXIinactivation was first-order in all conditions examined. Theenergy of activation for irreversible inactivation was alsostrongly influenced by the metal present, ranging from342 kJ·mol 2 1 (apoenzyme) to 604 kJ·mol 2 1 (Mg 2 þ –enzyme) to 1166 kJ·mol 2 1 (Co 2 þ –enzyme). These resultssuggest that the first irreversible event in BLXI unfolding isthe release of one or both of its metals from the active site.Although N  þ  Q content was an indicator of thermo-stability for class II XIs, this pattern may not hold for othersets of homologous enzymes. In fact, the extremelythermostable  a -amylase from  B. licheniformis  was foundto have an average N  þ  Q content compared withhomologous enzymes from a variety of mesophilic andthermophilic sources. Thus, it would appear that proteinthermostability is a function of more complex moleculardeterminants than amino-acid content alone. Keywords :  Bacillus licheniformis ; metal binding; thermo-stability; xylose isomerase.It has become apparent that protein thermostability arisesnot from a single chemical or physical factor, but fromnumerous subtle contributions integrated over the entiremolecular structure [1–6]. Thermostable proteins usuallyexhibit no significant differences in backbone conformationwhen compared with less thermostable proteins, but theytypically have increased numbers of salt bridges, sidechain–side chain hydrogen bonds, and residues involved in a  helices [7–9]. Stability at very high temperatures furtherrequires that a particular enzyme resist thermally induceddeleterious chemical reactions, which usually occur atinsignificant rates at lower temperatures [10]. For example,one of the most evident patterns in the amino-acidcomposition of hyperthermophilic proteins is the biasagainst thermally labile amino-acid residues. This pattern isobvious on examination of the amino-acid compositions of the total protein content of eight mesophilic and sevenhyperthermophilic micro-organisms for which genomesequence data are available (Table 1). The most strikingdifference is the 55% reduction in the number of glutaminesin hyperthermophilic proteins; note also the 28% reductionin asparagines. As these two amino acids are easilydeamidated at elevated temperatures [10–13], it is notsurprising that they are less abundant in proteins fromhyperthermophiles. This observation raises the question of whether a relatively low N  þ  Q content is a signature of enhanced thermostability in proteins from mesophilicsources.The potential use of biocatalysts at high temperatures fortechnological purposes has drawn interest in developingthermoactive and thermostable enzymes that would providesignificant processing advantages [14]. For example,thermostable xylose isomerases (XIs) (EC 5.3.1.5), whichcatalyze the isomerization of   D -xylose to  D -xylulose  invivo ,are used for the conversion of   D -glucose into  D -fructose forthe production of high-fructose corn syrup [15]. Elevatedbioprocessing temperatures are preferred to achieve highercatalytic rates as well as more favorable equilibrium yields Correspondence to  J. G. Zeikus, Department of Biochemistry andMolecular Biology, 410 Biochemistry Building, Michigan StateUniversity, East Lansing, MI 48824, USA. Fax:  þ  517 353 9334,Tel.:  þ  517 353 5556, E-mail: zeikus@msu.edu(Received 24 April 2001, revised 29 September 2001, accepted10 October 2001)  Abbreviations : XI, xylose isomerase; BLXI,  Bacillus licheniformis xylose isomerase; DSC, differential scanning calorimetry. Eur. J. Biochem.  268,  6291–6301 (2001) q FEBS 2001  [16]. Because of the commercial importance of XIs, theirbiochemical, biophysical, and structural properties havebeen extensively studied, and abundant sequence infor-mation is available [10,17–24]. On the basis of the absenceor presence of a 50-residue insert at the N-terminus, XIshave been classified into class I and class II enzymes,respectively [25]. Two distinct metal-binding sites, M1 andM2, have been identified in all XIs: (a) the metal in site M1is co-ordinated to four carboxylate groups; (b) the metal insite M2 is co-ordinated to one imidazole and threecarboxylate groups. The metals in sites M1 and M2 wereinitially referred to as structural and catalytic metals,respectively [18,26,27], but these appellations are no longervalid, because later studies showed that both metals aredirectly involved in catalysis [24,28,29]. The stabilizing andactivating metals are typically the bivalent cations Mg 2 þ ,Co 2 þ , and Mn 2 þ . Metal specificity depends on both thenature of the substrate (i.e. glucose or xylose) and whetherthe enzyme is a class I or class II XI.  Thermus aquaticus  XI,a class I enzyme, isomerizes glucose most efficientlywhen in the presence of Mn 2 þ , but its activity towardxylose is highest with Co 2 þ as the cofactor [26]. The class II  Bacillus coagulans  XI, on the other hand, isomerizesxylose most efficiently when in the presence of Mn 2 þ ,whereas its activity toward fructose is best promoted byCo 2 þ [27].Class I XIs are a relatively homogeneous group when itcomes to thermostability, whereas class II XIs vary widelyin this regard. This heterogeneity among class II XIs mayarise from the existence of additional salt bridge(s) specificto thermostable class II XIs [30]. In a previous study of classII XIs, a positive correlation between the enzyme’s N þ Qcontent and the growth temperature of the source organismwas observed: XIs from the most thermophilic sourcestypically had lower N þ Q content [23]. Of the XIsexamined, only the enzyme from the mesophilic bacterium  Bacillus licheniformis  DSM13 (BLXI) was atypical.Originating from an organism that optimally grows at37 8 C, BLXI contains only 26 N þ Q residues, comparedwith 23 in the enzyme from the hyperthermophile Thermotoga neapolitana  (optimal growth at 80 8 C), and46 in the XI from the mesophile  Escherichia coli  (optimalgrowth at 37  8 C). The low Q (and, to some extent, N)content in proteins from hyperthermophiles (Table 1) raisesan interesting question: does a relatively low N þ Q contentin a protein from a mesophile indicate an unusually highthermostability for this protein? The location of N and Qresidues within a protein structure is certainly a criticalconsideration, but detailed structural information is oftennot available to make this determination. In an attempt totest the simple hypothesis that class II XI stability at hightemperatures correlates with its N þ Q content, independentof the growth temperature of the source organism, thebiochemical and biophysical properties of the previouslyuncharacterized BLXI were determined. Particular empha-siswas placedon theinfluence ofbivalent cations on activityand stability. Our results show that for class II XIs, N þ Qcontent relates to the enzyme’s functional temperature rangeand that BLXI thermostability is also directly related to thebinding of specific metals as cofactors. At the same time, thesimple relationship between thermostability and N þ Qcontent may not hold in general, as it is not the case for thethermostable  a -amylase from  B. licheniformis . MATERIALS AND METHODS B. licheniformis xylA  gene cloning  B. licheniformis  strain DSM13 was grown at 37 8 C inLuria–Bertani broth [31]. A  B. licheniformis  genomic DNAlibrary was constructed in vector pUC18 (Pharmacia,Piscataway, NJ, USA), using methods described in [23]. E. coli  xyl – mutant HB101 (F – ,  hsdS20, ara-1, recA13, proA12, lacY1, galK2, rpsL20, mtl-1, xyl-5 ) [32] wastransformed with the ligation mixture by electroporation andplated on M9 medium containing 0.2% xylose, 0.1%casamino acids, thiamine (500  m g·mL 2 1 ), and ampicillin(100  m g·mL 2 1 ). Only the transformants expressing arecombinant XI produced large colonies on this medium. Enzyme purification BLXI was purified from a 2-L culture of   E. coli  HB101carrying plasmid pBL2 grown in M9 medium (comple-mented as above). After centrifugation for 5 min at 4000  g ,the cell pellet was resuspended in 50 m M  Mops (pH 7.0)containing 5 m M  MgSO 4  and 0.5 m M  CoCl 2  (buffer A). Thecells were disrupted by two consecutive passes through aFrench pressure cell (American Instrument Co., SilverSpring, MD, USA), using a decrease in pressure of 96.5 MPa. After centrifugation at 25 000  g  for 30 min, thesupernatant was heat-treated at 60 8 C for 10 min. Theprecipitated material was separated by centrifugation at25 000  g  for 30 min. The soluble fraction was loaded on toa DEAE–Sepharose Fast-Flow column equilibrated withbuffer A. The protein was eluted with a linear 0.05–0.4  M NaCl gradient in buffer A. The active fractions wereanalyzed by SDS/PAGE (12% acrylamide) and stained withCoomassie blue R250. The homogeneous fractions werepooled and extensively dialyzed against buffer A. Proteinconcentrations were determined using the Bio-Rad proteinassay kit (Bio-Rad, Richmond, CA, USA), with BSA as thestandard. The purified enzyme was stored at  2 70 8 C untiluse. Molecular mass determination BLXI molecular mass was determined by gel filtration usinga Sephacryl S-300 HR column (1.4 cm  £  160 cm)calibrated with blue dextran and protein standards of 443,200, 150, and 66 kDa (Sigma Chemical Co., St Louis, MO,USA). The flow rate was 0.2 mL·min 2 1 . EDTA treatment The purified enzyme was incubated overnight at 4 8 C inbuffer A containing 10 m M  EDTA. It was then dialyzedtwice against 50 m M  Mops (pH 7.0) (SigmaUltra, SigmaChemical Co.) containing 2 m M  EDTA, and finally dialyzedtwice against 50 m M  Mops (pH 7.0), this time withoutEDTA. The apoenzyme was divided into aliquots and storedat 2 70 8 C until use. Enzyme assays BLXI activity was assayed routinely with glucose as thesubstrate. The enzyme (0.06 mg·mL 2 1 ) was incubated in q FEBS 2001  B. licheniformis  xylose isomerase ( Eur. J. Biochem. 268 ) 6293  50 m M  Mops (pH 7.0 at room temperature) containing1 m M  CoCl 2  and 1  M  glucose at 60 8 C for 20 min Thereaction was stopped by transferring the tubes to an ice bath.The amount of fructose produced was determined by thecysteine/carbazole/sulfuric acid method [33]. To determinethe effect of temperature on BLXI activity, the holo-BLXIwas incubated in the reaction mixture at the temperatures of interest in a Perkin–Elmer Cetus GeneAmp PCR system9600 (Perkin–Elmer, Norwalk, CT, USA) for 20 min. Todetermine the kinetic parameters, assays were performed inthe presence of either 80–1400 m M  glucose or 20–900 m M xylose.The amounts offructose and xylulose produced weredetermined using the cysteine/carbazole/sulfuric acidmethod. Absorbance was measured at 537 nm and 560 nmfor xylulose and fructose, respectively. One unit of isomerase activity is defined as the amount of enzyme thatproduces 1  m mol of product per min under the assayconditions. Activation of apo-BLXI by metals To examine the effect of bivalent cations on enzyme activity,CoCl 2 , MnCl 2 , or MgCl 2  were added to the apoenzymereaction mixture at concentrations of 0.003–100 m M (activity on glucose) or 0.002–0.03 m M  (activity onxylose). Activity on xylose was determined using0.024 mg·mL 2 1 apo-BLXI and 635 m M  xylose. Activityon glucose was determined using 0.06 mg·mL 2 1 apo-BLXIand 1  M  glucose. The activation constant,  K  act , for a metal isdefined as the metal concentration that results in 50% of maximum activity [34,35]. Enzyme thermoinactivation The apo-BLXI (0.17 mg·mL 2 1 ) in 50 m M  Mops (pH 7.0 atroom temperature) was incubated at various temperatures(in a Perkin–Elmer Cetus GeneAmp PCR system 9600) fordifferent periods of time. Thermoinactivation was stoppedbytransferring the tubes to an ice bath. Residual activity wasdetermined under the conditions described above, exceptthat the reaction mixture contained 5 m M  CoCl 2  instead of 1 m M  CoCl 2 . To determine the effect of metals on theapoenzyme stability, 0.5 m M  CoCl 2 , 0.5 m M  MnCl 2 , or2 m M  MgCl 2  was added to the enzyme solution. Theresulting solution was equilibrated for 30 min at 30 8 Cbefore thermoinactivation was initiated (preincubationconditions known to be sufficient for the metal to reachequilibrium between the buffer and enzyme metal-bindingsites [19]). Heat-induced enzyme precipitation Heat-induced enzyme precipitation was monitored from25 8 C to 90  8 C by light scattering ( l  ¼  580 nm), using theapo-BLXI (0.08 mg·mL 2 1 ) in 10 m M  Mops (pH 7.0).Absorbance measurements were conducted in 0.3-mLquartz cuvettes (pathlength 1 cm) using a BeckmanDU-650 spectrophotometer equipped with a Peltiercuvette-heating system. The increasing thermal gradientwas 1.0 8 C·min 2 1 . The effect of metals on apo-BLXIprecipitation was studied in the presence of 0.5 m M  CoCl 2 ,0.5 m M  MnCl 2 , or 5 m M  MgCl 2 . PH studies The effect of pH on BLXI activity was determined at 64 8 Cusing the routine assay described above, except that theMops buffer was substituted with 100 m M  sodium acetate(pH 4.0–5.7), 100 m M  Pipes (pH 6.0–7.5), or 100 m M Hepps (pH 7.5–8.7). All pHs were adjusted at roomtemperature, and the  D p K  a  /  D T   values for acetate, Pipes, andHepes (0.000,  2 0.0085, and  2 0.011, respectively) [36]were taken into account for the results. Differential scanning calorimetry (DSC) DSC experiments were performed on a Nano-Cal differen-tial scanning calorimeter (Calorimetry Sciences Corp.,Provo, UT, USA) using a scan rate of 1 8 C·min 2 1 . Sampleswere scanned from 25 8 C to 100 8 C. The apoenzyme(obtained by EDTA treatment, see above) was scannedagainst 50 m M  Mops (pH 7.0). For the metal-containingenzymes, the apoenzyme was incubated for 2 h at roomtemperaturewith 5 m M  metal chloride. The enzyme solutionwas then dialyzed once against 1 L 50 m M  Mops (pH 7.0)to remove the metal that was not tightly bound to theenzyme. Each metal-containing enzyme was scannedagainst the corresponding dialysis buffer. The dialysisbuffer was used to generate the baseline. The enzymecontaining both Mg 2 þ and Co 2 þ was dialyzed against bufferA, then scanned against the dialysis buffer as control. RESULTS AND DISCUSSION Cloning of the  B. licheniformis xylA  gene Plasmid pBL1 was characterized from an HB101 transfor-mant that formed large colonies on M9 medium containingxylose. Comparison of the physical map of the plasmidpBL1 insert with that of plasmid pWH1450 [37] indicated Fig. 1. Determination of BLXI molecular mass by gel filtration. V  e  /V  o  is the ratio of a protein’s elution volume to the elution volume of blue dextran. ( X ) Protein standards; ( A ) BLXI. Linear regression (1),with an  r   2 of 0.951, is based on the elution data of all four proteinstandards. Linear regression (2), with an  r  2 of 0.981, is based on theelution data of the 200, 150, and 66 kDa protein standards. Linearregressions (1) and (2) give molecular masses of 200 and 177.5 kDa,respectively, for BLXI. 6294 C. Vieille  et al . ( Eur. J. Biochem. 268 )  q FEBS 2001  that pBL1 contained  B. licheniformis xylR ,  xylA , and atruncated  xylB.  A 1.2-kb  Sph I– Eco RI fragment was deletedfrom the pBL1 insert to inactivate the  B. licheniformis xylR repressor gene, leading to plasmid pBL2. Plasmid pBL2 wasused to express BLXI in the rest of the study. Purification of the recombinant protein and physicalproperties The  B. licheniformis xylA  gene was expressed from its ownpromoter in plasmid pBL2. The recombinant enzyme waspurified (heat treatment plus DEAE–Sepharose chromato-graphy) from an HB101 (pBL2) 2-L culture grown in M9medium plus xylose. The purified enzyme was shown to behomogeneous by SDS/PAGE and staining with Coomassieblue. Approximately 100 mg enzyme was obtained from the2-L culture. A molecular mass of 200 kDa for the nativeprotein was estimated by gel filtration (Fig. 1). Analysis bySDS/PAGE showed a single band with a molecular mass of about 50 kDa. This estimate is in agreement with thatexpected from the protein sequence (50 905 Da). Theseresults indicate that BLXI is expressed as a homotetramerin  E. coli . It is interesting to note that XIs from thethermophile  Thermoanaerobacterium thermosulfurigenes and the hyperthermophile  T. neapolitana , both homotetra-mers in their native forms, are expressed as active dimersin  E. coli  [38]. Effects of temperature and pH on BLXI activity The effect of temperature on BLXI activity was determinedby measuring the holoenzyme activity on glucose in thepresence of 1 m M  CoCl 2 . As shown in Fig. 2A, BLXI isoptimally activebetween 70 8 C and 72  8 C. Above72  8 C, theenzyme rapidly loses activity,and itis completely inactiveat80  8 C. The Arrhenius plot for BLXI activity is linearbetween 35  8 C and 66  8 C. The estimated energy of activation ( E  a ) for BLXI activity is 76 kJ·mol 2 1 , an  E  a value comparable to those for  E. coli  and  T. neapolitana  XIactivities (70 and 80 kJ·mol 2 1 , respectively; unpublisheddata).The effect of pH on BLXI activity was determined bymeasuring the holoenzyme activity on glucose betweenpH 4.8 and 8.2 (values after temperature correction). BLXIis optimally active at pH 7.2 and shows more than 80%activity between pH 6.8 and 7.6 (Fig. 2B). BLXI kinetic parameters BLXI kinetic parameters were determined at 60 8 C forglucose and xylose using the holoenzyme in the presence of 1 m M  CoCl 2  (Table 2). Not surprisingly, BLXI is about sixtimes more efficient on xylose than on glucose, as indicatedby the values of   V  max  /  K  m . Compared with other type IIthermophilic XIs (Table 2), BLXI has average kineticparameters, but it has a relatively low catalytic efficiency onxylose. BLXI thermostability and inactivation characteristics BLXI thermostability was first characterized using theholoenzyme in the presence of 1 m M  CoCl 2 . In theseconditions, BLXI has a half-life of 14 h at 64  8 C, 70 min at67  8 C, and 2 min 40 s at 70 8 C. Figure 3 shows that BLXIinactivation at 68  8 C was first-order. Inactivation ratesobtained for three different enzyme concentrations at 68 8 C Table 2. Kinetic constants of thermophilic type II XIs. Organism T  ( 8 C)Glucose XyloseReference V  max (U·mg 2 1 ) K  m (m M )  V  max  /  K  m V  max (U·mg 2 1 )  V  max  /  K  m K  m (m M )  B. licheniformis  DSM13 60 7.7 145 0.053 22.2 67 0.33 This work   B. stearothermophilus  60 6.0 220 0.027 44.5 100 0.44 [55] T. saccharolyticum  B6A 65 6.3 120 0.053 17.6 16 1.10 [56] T. thermosulfurigenes  4B 65 5.3 142 0.037 15.7 20 0.78 [56] T. maritima  DSM 3109 90 16.2 118 0.137 68.4 74 0.92 [57] T. neapolitana  5068 90 22.4 89 0.253 52.2 16 3.28 [23] Fig. 2. Effects of temperature and pH on BLXIspecific activity.  (A) Arrhenius plot of BLXIspecific activity as a function of temperature. Thelinear regression was only applied to thetemperature points below the optimumtemperature for activity. (B) Effect of pH on BLXIactivity. Assays were performed at 64 8 C in100 m M  sodium acetate ( A ; pH 4.0–5.7), 100 m M Pipes ( X ; pH 6.0–7.5), or 100 m M  Hepes ( K pH 7.5–8.7). The  D p K  a  /  D T   values for acetate,Pipes, and Hepps were taken into account tocalculate the pH values at 64 8 C. All assays wereperformed in triplicate. q FEBS 2001  B. licheniformis  xylose isomerase ( Eur. J. Biochem. 268 ) 6295
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