Báo cáo Y học: Stabilization of a (ba)8-barrel protein by an engineered disulfide bridge potx

Stabilization of a (ba) 8 -barrel protein by an engineered disulfide bridge Andreas Ivens 1 , Olga Mayans 2 , Halina Szadkowski 3 , Catharina Ju¨ rgens 1 , Matthias Wilmanns 2 and Kasper Kirschner 3 1 Universita ¨ tzuKo ¨ ln, Institut fu ¨ r Biochemie, Ko ¨ ln, Germany; 2 EMBL c/o DESY, Hamburg, Germany; 3 Biozentrum, Universita ¨ t Basel, Basel, Switzerland The aim of this study was to increase the stability of the thermolabile ( ba) 8 -barrel e nzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disul- fide bridges. For the de sign o f s uch v ariants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds. These variants avoid short-range connections, substitutions near catalytic residues, and crosslinks between the new and t he three parental cysteines. The variant linking residues 3 and 189 fastens t he N-terminus to the (ba) 8 -barrel. The rate of ther- mal i nactivation at 50 °C of this variant with a closed disulfide bridge is 65-fold slower than th at of the r eference dithiol form, but only 13-fold slower than that of the parental protein. The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman’s reagent as well as the decreased turnover number indicate that the protein structure i s rigidified. T o confirm these data, we have solved the X-ray structure to 2 .1-A ˚ resolution. The second variant was designed to crosslink the terminal modules ba1 and ba8. However, not even the d ithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein. Keywords: indoleglycerol phosphate synthase; (b/a) 8 -barrel proteins; stabilizing disulfide bonds; protein e ngineering. Indoleglycerol phosphate synthase (IGPS) is a (ba) 8 -barrel protein with an N-terminal extension of 48 residues. In Escherichia coli, IGPS (eIGPS) is the N-terminal domain of a monomeric, bifunctional enzyme, where the C-terminal domain is phosphoribosyl anthranilate isomerase (ePRAI), folded into another (ba) 8 -barrel [1]. The catalytic efficiencies of the engineered separated domains are virtually identical to those in the bifunctional enzyme [2]. eIGPS is, however, more labile than ePRAI. The catalytic activity of eIGPS decays at 55 °C w ith a half-life o f 0 .5 min [3]. In contrast, ePRAI activity decays at 60 °C with a half-life of 100 min (R. Sterner, Institut fu ¨ r Biochemie, Universita ¨ tzuKo ¨ ln, Germany, personal communication). The eIGPS domain, in turn, is also more labile than eIGPS in the native bifunctional protein [4,5,6]. In contrast to eIGPS [1], the IGP synthases from the hyperthermophiles Sulfolobus solfataricus (sIGPS [7]) and Thermotoga maritima (tIGPS [3]), are thermostable, mono- functional monomers. The comparison of the three high resolution crystal structures suggests that an increased number of salt bridges over that in eIGPS decreases the rates of irreversible thermal inactivation of both sIGPS and tIGPS. In support o f this proposal, mutational disruption of salt bridge that crosslinks its terminal ba1andba8 modules, significantly destabilized the variants b y comparison to the parental enzyme [3], in support of analogous findings reported previously [8]. The aim of this work was to stabilize the labile eIGPS domain by introducing new disulfide bonds rather than new salt bridges. Disulfide bonds can stabilize proteins under- going reversible unfolding by decreasing the main chain entropy of their unfolded states [9–11]. For example, the most thermostable single-disulfide variants of the mono- meric xylanase were crosslinked between the N- and C-termini [12]. Similar observations have been made with another monomeric (ba) 8 -barrel protein, PRAI from yeast (yPRAI [13]). Disulfide bonds can also stabilize irreversibly unfolding proteins by decreasing the unfolding rate [14,15]. This investigation focuses on two variants of the eIGPS domain that are predicted to form geometrically favourable single, new, long-range disulfide bonds, far removed from the a ctive site and the parental cyste ines. The y either clamp the N-terminus to the core of the (ba) 8 -barrel fold or crosslink the ba1andba8 modules. MATERIALS AND METHODS DNA manipulations and sequence analysis Preparation o f DNA samples, digestion with restriction endonucleases, agarose gel electrophoresis, and DNA Correspondence to A. Ivens, Universita ¨ tzuKo ¨ ln, Institut fu ¨ r Biochemie, Otto-Fischer-Str. 12–14, D-50674 Ko ¨ ln, Germany. Fax: +49 221 4706 731, Tel.: +49 221 470539, E-mail: andreas.ivens@uni-koeln.de Abbreviations: IGP, indoleglycerol phosphate; CdRP, 1-(o-carboxy- phenylamino)-1-deoxy- D -ribulose-5-phosphate; PRA, N-phos- phoribosyl anthranilate; ePRAI, PRA isomerase domain from Escherichia coli; eIGPS, IGP synthase domain from Escherichia coli; eIGPS-PRAI, indoleglycerol phosphate synthase–phosphoribosyl- anthranilate isomerase bifunctional protein from Escherichia coli; etrpC, gene encoding eIGPS; sIGPS, IGP synthase from Su lfolobus solfataricus; tIGPS, IGP synthase from Thermotoga maritim a;Nbs 2 , 5,5¢-dithiobis(2-nitrobenzoic acid); ASA, accessible surface area. (Received 30 August 2001, revised 30 November 2001, accepted 17 December 2001) Eur. J. Biochem. 269, 1145–1153 (2002) Ó FEBS 2002 ligation were performed as described by Sambrook et al . [16]. Pulsed liquid-phase sequencing was carried out on a Applied Biosystems 477 A sequencer according to the manufacturer’s specifications. PCRs were performed in the Trio-block from Biometra (Go ¨ ttingen, Germany), using thermostable Pyrococcus furiosus DNA-polymerase (Stratagene, Heidelberg, Germany). Oligonucleotides were purchased from Microsyn (Windisch, Switzerland). Strains and plasmids Protein was expressed in E. coli BL21(DE3) [F – ompT gal [dcm] [Ion] hsdS B (r B – m B – )], genetic manipulation and mutagenesis was carried out with E. coli JM109 [F¢,traD36 lacI q D(lacZ)M15 p roA + B + /e14 – (McrA – ) D(lac-proAB) thi gyrA96(Nal r ) endA1 hsdR17 (r K – m K + ) relA1 supE44 recA1]. The expression vector pET21a(+), where protein production is under control of the T7-RNA-polymerase promoter [17], was used for expression of etrpC mutants. Oligonucleotides The following PCR primers were used t o amplify the etrpC gene from the vector pMc-C/F, which contains the bifunc- tional eIGPS:ePRAI gene [2]. The 5¢ primer was used as a mutagenic primer for replacing Thr3 by Cys (bold letters indicate the mutated codon). T3C 5¢ primer, 5 ¢-CGAGGG TAA CATATGCAATGCGTTTTAGCGAA-3¢;etrpC3¢ primer, 5¢-CCACGCGTC AAGCTTCATACTTTATTC-3¢. The NdeIandHindIII restriction sites for ligation into vector pET21a(+) are underlined, respectively. The following primers were used for replacing Arg189 by Cys: T189C 5 ¢ primer, 5¢-AACCGTGATCTGTGCGATT TGTCGATT-3¢; R189C 3 ¢ primer, 5¢-AATCGACAAATC GCACAGATCACGGTT-3¢. The mutated codons are showninboldletters. The following four primers were used for replacing both Ile64 and Met240 by Cys: I64C 5¢ primer, 5¢-AAAG GCGTGTGTCGTGATGATTTCGATCCA-3¢;I64C3¢ primer, 5¢-GAAATCATCACGACACACGCCTTTTGA 3¢; M240C 5¢ primer, 5¢-GCGTTGTGTGCCCATGACGAT TTG-3¢; M240C 3¢ primer, 5 ¢-ATCGTCATGGGCAC ACAACGCCGAACCAAT-3¢. The mutated codons are shown in bold letters. The 5 ¢ prim er, which introduced an NdeI site (underlined) into the etrpC gene for eIGPS (64–240) was: etrpC5¢primer, 5¢-ACGAGGGTAA CATA TGCAAACCGTTTTAGC-3¢. Universal T7 p romoter and terminator primers were used for sequencing (Novagen). Both primers anneal to the T7 promoter and terminator sequences in pET21a(+), up- and downstream of the etrpCgene. PCR and site-directed mutagenesis The v ector pMc2- trpC/F [2] was u sed as the template for the production of the double mutant eIGPS(3–189) and eIGPS(64–240). Site-directed mutagenesis was performed by PCR with the overlap-extension method [18,19]. The PCR mix c ontained 250 l M of each nucleotide triphos- phate, 20 pmol of each primer, 0.1 lg of t emplate, 4 lLof 10 x Pfu reaction buffer and 2.5 U of Pfu -DNA polymerase (Stratagene) in a total volume of 40 lL. The amplification protocol for the production of megaprimers consisted of 3 min at 95 °C, followed by 35 cycles of 1 min at 95 °C, 2 min at 55 °C and 3 min at 72 °C. The megaprimers were purified by electrophoresis on a 0.8% agarose gel. They were used as templates in various dilutions and a t a reduced annealing t emperature of 50 °C in a second PCR r eaction with the 5 ¢ and 3¢ primers to yield the full-length mutated gene. The resulting etrpC fragment was purified by electro- phoresis on a 0 .8% agarose gel, digested with NdeIand HindIII and purifi ed again. The fragment was then ligated into a NdeI–HindIII digested and dephosphorylated pET21a(+) vector, yielding the vector pET21a(+)-etrpC. After transformation of E. coli BL21(DE3) with pET21a(+)-etrpC, transformants w ere grown overnight in 2 m L Luria–Bertani medium [16], containing 0.1 lg amp- icillinÆmL )1 (Luria–Bertani/amp medium). The plasmids were isolated and digested with NdeIandHindIII to screen for clones with inserts. One positive clone was confirmed by complete DNA sequencing. Expression and purification of E. coli eIGPS(3–189) The protein was expressed in E. coli BL21(DE3). Single colonies harbouring the plasmid pET21a(+)-etrpC (3–189) were grown overnight in Luria–Bertani medium [16], supplemented w ith 100 lgÆmL )1 ampicillin at 37 °C. On the following day, 15 L Luria–Bertani/amp medium in conical flasks were inoculated with 15 mL of the overnight culture. The cells were allowed to grow for three days at 22 °C. After 24 and 48 h, 50 lgÆmL )1 ampicillin was again added. The cells were harvested a fter 64 h and suspended in 50 m M potassium phosphate, pH 7.8, with 1 m M EDTA. For breakage of the cells, the suspension was sonified in a Branson sonifier (2 · 2 min, level 5, 60% pulse, ice cooled). DNase and RNase were added to a final c oncentration of 5 lgÆmL )1 for digestion of nucleic acids. The homogenate was centrifuged twice at 10 000 g,4°C and the supernatan t was diluted with d eionized water to yield a conductivity of 1.27 mS Æcm )1 . The crude extract was loaded on a DEAE–Sepharose fast-flow column (5 · 25 cm, 510 mL) with a flow rate of 205 m LÆh )1 , equilibrated with 10 m M potassium phosphate, pH 7.5. After washing with equilibration buffer for 1.5 col- umn vol., the column was eluted with a linear gradient from 10 to 300 m M potassium phosphate buffer pH 7.5, 1 m M EDTA. eIGPS(3–189) eluted, as determined by activity and SDS/PAGE, at a phosphate concentration of 150 m M . Fractions containing eIGPS(3–189) were pooled and dialyzed overnight against 5 m M potassium phosphate buffer pH 6.8, 100 m M KCl. The dialysate was loaded onto a hydroxylapatite column (2.5 · 25 cm, 122 mL) with 34.2 mLÆh )1 , that had been equilibrated w ith 5 m M potassium phosphate buffer pH 6.8, 100 m M KCl, washedwith 1 column vol. at 68.2 mLÆh )1 and eluted w ith a linear gradient from 5 to 300 m M potassium phosphate buffer pH 6.8, 100 m M KCl. At the same flow rate, eIGPS(3–189) eluted at 1 00 m M potassium phosphate pH 6.8, 100 m M KCl. Fractions containing eIGPS(3–189) were pooled and concentrated by ultrafiltration to 10 mg ÆmL )1 for a gel permeation chromatography run. The concentrated protein solution was adjusted to a final concentration of 300 m M NaCl, 3% s ucrose (v/v). The solution was loaded on a Sephacryl S-200 column (2.5 · 90 cm, 440 mL) equilibrate d with 50 m M potassium 1146 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002 phosphate buffer pH 7.5, 300 m M NaCl, and eluted with equilibration buffer at a flow rate o f 34.2 mLÆh )1 . Fractions with pure eIGPS(3–189) were concentrated by ultrafiltration andstoredat)70 °C after dripping into liquid nitrogen. Enzymatic assay for indoleglycerol phosphate synthase Indoleglycerol phosphate synthase activity was assayed at 25 °Cin50m M Tris/HCl pH 7.5, 1 m M EDTA, with 40– 70 n M eIGPS and 3–5 l M CdRP. The reaction was started by addition of the nonfluorescent substrate C dRP [ 20]. Appearance of IGP was measured continuously by its fluorescence excited at 280 nm a nd emitted a t 350 nm. Because IGP accumulates, t he progress curves were fitted to the integrated M ichaelis–Menten equation that takes com- petitive product inhibition into account [21]. The formation of 1 lmol IGP per minute at 25 °C was defined as one unit of activity (Table 1). SDS/PAGE SDS/PAGE was carried out according to the method of Laemmli [22]. The stacking gel and separation gel contained 6 and 12.5% acrylamide, respectively. The protein samples were mixed with 1 vol. of 2 x SDS-sample buffer ( 100 m M Tris, pH 6.8, 1% SDS, 20% glycerol, 0.01% bromphenol blue) and heated to 100 °C for 5 min before loading. The gels were run with constant current of 30 mA for 1–2 h, stained with Coomassie Brilliant Blue solution (0.1% Coomassie blue, 20% acetic acid, 40% methanol) and destained by boiling in water for 5 min in a microwave oven. Proteins used as molecular mass standards were bovine pancreatic trypsin inhibitor ( 6.5 kDa), myoglobin (16.9 kDa), E. coli phosphoribosyl anthranilate isomerase (21.1 kDa), E. coli a-tryptophan synthase (28.7 kDa), indoleglycerol phosphate synthase (31 kDa), E. coli b-tryptophan synthase (43 kDa), BSA (66.3 kDa) and phosphorylase b (97.4 kDa). Protein concentrations were determined according to Bradford [23] with known concentrations of BSA as standard, as well as with absorbance spectroscopy at k ¼ 280 nm (e eIGPS ¼ 0.81 cm 2 Æmg )1 ) in a Hewlett Packard Diode Array spectrophotometer (model 8452 A), connected to a HP Vectra ES/12 computer. Protein thermal stability determined by inactivation kinetics For stability measuremen ts, the enzymes we re incubated in 0.1 M potassium phosphate buffer at a given temperature and irreversibly heat inactivated. Aliquots were taken at certain time points and chilled on ice, until the remaining activity was determined (in Tris, as described above) and plotted against the incubation time. Kinetic data were obtained a s d escribed above. Incubation buffer was 100 m M potassium phosphate, pH 7.5, 1 m M EDTA, 1 m M dithio- threitol. D ithiothreitol was omitted in the case of oxidized eIGPS(3–189). Oxidation and reduction of the engineered disulfide bridge in eIGPS(3–189) For reduction of the disulfide bridge, the enzyme at a concentration o f 1.5 mgÆmL )1 was i ncubated f or 6 h at 4 °C in 50 m M potassium phosphate pH 7.5, 300 m M NaCL, 10 m M dithiothreitol. For promoting the formation of the disulfide bond, the enzyme was incubated overnight at 4 °C in 50 m M potassium phosphate pH 7.5, 300 m M NaCL, supplemented with 0 .5 m M Nbs 2 as the o xidizing com- pound. To examine whether the thiols are reduced or the disulfide bridge is formed, the protein samples were run on a nonreducing SDS/PAGE. Determination of thiol content The content of free S H groups and cysteines involved in a disulfide bridge was d etermined according t o the reaction of Ellmann [24]. Stock solutions of assay buffer were 1 m M Nbs 2 in 50 m M Na-phosphate buffer pH 7.5, 1 m M EDTA or 50 m M Tris pH 7.5, 1 m M EDTA. The following extinction coefficients were used: e TNB (440 nm) ¼ 9.22 m M )1 Æcm )1 , eNbs 2 (325 nm) ¼ 17.38 m M )1 Æcm )1 . Excess amounts of both re ducing and oxidizing compounds were removed before the measurements by gel filtration on NAP columns (Pharmacia). A blank run was performed with assay buffer before the protein was added to a final concentration of 10–30 l M in a final volume of 1 mL. After various time points the absorption at 440 nm was recorded. CD spectra CD spectra were monitored with a Jasco model J -720 spectropolarimeter, which was connected to a Philips SX computer. The measurements were carried out in 0.05 M Na-phosphate buffer, pH 7.5, 1 m M EDTA in the absence of oxidizing agent for oxidized forms and in the presence o f 1m M dithiothreitol for reduced forms. For all CD measurements, 10 spectra were recorded and averaged. X-ray structure solution Prior to crystallization, the protein buffer was exchanged in NAP 10 columns (Pharmacia) to 50 m M potassium phos- phate,pH7.5,1m M EDTA. The protein was then concentrated to 10 mgÆmL )1 in Centriprep and Centricon ultrafiltration units (Amicon). Crystallization was carried Table 1. Purification of the eIGPS(3–189) disulfide variant from 69 g (wet weight) of transformed E. coli cells. Fraction Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Yield % Crude extract 2093 529 0.3 100 Anion exchange eluate 311 313 1.0 59 Hydroxylapatite eluate 208 208 1.0 40 Gel filtration eluate 102 239 2.3 45 Ó FEBS 2002 Stabilization of a (ba) 8 -barrel protein (Eur. J. Biochem. 269) 1147 out as reported for the wild-type monomeric eIGPS [25] in 50 m M potassium phosphate, pH 5 .0, 1.2 M ammonium sulphate and 5 m M EDTA. Data were collected at the synchrotron radiation beam line X11 (EMBL c/o DESY, Hamburg) from shock-frozen crystals at 100 K using 30% (v/v) glycerol as cryoprotec- tant. Data were recorded on a MAR-CCD detector in three resolution sweeps to a maximum resolution of 2.1 A ˚ . The crystals belong to the space group P6 3 22 and are affected by strong pseudosymmetry. A large cell with dimensions a ¼ 141.4 A ˚ 2 , c ¼ 156.7 A ˚ and containing three molecules per asymmetric unit coexists with a subcell of dimensions a ¼ b ¼ 81.6 A ˚ 2 , c ¼ 156.7 A ˚ and one molecule per asymmetric unit. Both cells are related b y a rotation of 30° around c. Only reflections corresponding to the subcell (k ¼ h ±3n) show significant intensities, with reflections from the larger cell being remarkably weaker. This crystallo - graphic problem also affected monomeric, wild-type eIGPS and has been described previously [25]. The structure presented here corresponds to that of the subcell and therefore represents an averaged model. The HKL suite of programs [26] was used in data processing and reduction. The data set consisted of 17 306 unique reflections with a multiplicity of 8.8%, an overall R merge of 4.1% and a completeness of 93.7% (the outer resolution shell, 2.15–2.10 A ˚ , had values of 25.0% R merge , multiplicity 2.6% and 78% completeness). Structure solu- tion was carried out by the molecular r eplace ment technique (AMoRe [27]), using the eIGPS domain from the bifunc- tional enzyme [1] as a search model. For refinement, reflection data were divided into a working set and a t est set (1057 reflections) u sing FREERFLAG . Refinement was carried out using the CNS software [28] and included bulk solvent correction, overall anisotropic B-factor scaling and restrained, individual, isotropic B factor refinement. The structure has been refined to a crystallographic R-factor of 24.1% (R free 31.9%). The model includes protein residues 1–259, the CdRP compound and 187 solvent atoms. No obvious interpretable electron d ensity can b e observed for residues 1 and 2, so these were included as model s. The coordinates and structure factors have been depo- sited at the PDB with accession code 1218477 (1JCM). RESULTS AND DISCUSSION Design of disulfide bonds Engineering of a new disulfide bond into eIGPS must take into account the presence of three parental cysteines within the s trands b 1 (C54), b 3 (C113) and b 4 (C134) o f the ( ba) 8 barrel (Fig. 1). At first sight, any of these native cysteines might be used as disulfide-bonding partners. All three residues are, however, solvent-inaccessible: the A SA values, calculated with AREAIMOL [28a], are 1, 2 , and 0 A 2 , respectively. Although they are not conserved [1], and therefore n ot directly essential f or catalysis, C54 and C113 are nevertheless adjacent to two catalytically essential residues, namely E53 and K114 [5]. Initially, 12 potential residue pairs with an appropriate geometry for disulfide formation were identified, using the MODIP program [29]. Ten of these pairs were rejected, however, using the following criteria for exclusion: (a) pairs of residues le ading to short-ran ge d isulfides, that is, separated by less than 25 positions in the s equence; (b) positions adjacent to catalytic residues; a nd (c) new cysteines leading to geometrically favourable disulfides [29] with one of the three parental cysteines [30]. We avoided using the parental cysteines as partners for new disulfides, because the orientation of E53 and K114 might be altered by the introduction of an adjacent disulfide bridge, thus impairing catalysis. One of the preferred, new disulfide bonds requires the double substitution T3C/R189C, and fi xes t he N-terminus to the barrel core of this variant, designated eIGPS(3–189). The disulfide bond is accessible (ASA values of T3 and R189 are 54 and 68 A ˚ 2 , respectively) and fortuitously mimics one o f the e xtra salt bridges in both sIGPS [7] and tIGPS [3], which are missing in eIGPS [1]. However, the replacement R189C in eIGPS disrupts the parental short- range salt bridge of R189 to E169 on helix a5 . The other selected disulfide variant involves the double substitution I64C/M240C (ASA values o f I 64 and M240 are 27 and 0 A ˚ 2 , respectively), and is de signated eIGPS(64– 240). M240 is an invariant but solvent-inaccessible residue that anchors t he short h elix a8¢ to the core o f the protein [1]. This proposed disulfide crosslinks the loops b1a1andb8a8, which are widely separated i n sequence but adjacent i n space (Fig. 1 ), thus clamping the barrel between the N- and C-terminal modules ba 1 and ba 8 . This disulfide bond is topologically analogous to the strongly stabilizing disulfide bond introduced between helices a1anda8ofthemono- meric (ba) 8 -barrel protein yPRAI from yeast [13], and fortuitously mimics the s tabilizing salt bridge E73-R241 in tIGPS [3]. Production and purification of disulfide-bonded proteins The eIGPS variants ( 3–189) a nd (64–240) were produced by growing transformants of E. coli strain BL21 (DE3), as Fig. 1. Stereo representation of i ndoleglycerol phosphate synthase from E. coli. The bound phosphate ion indicates the location of the active site. The Ca positions of native cysteines (54, 113 a nd 134) and of the p lanned d isulfide bonds (3–189) and (64–240) are shown. 15, position of the single t ryptophan residue. 1148 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002 described in Materials and methods. As generally a larger fraction of soluble protein is expressed during c ell growth at temperature lower than at 37 °C [31], the cells were grown at 22 °C for 64 h. As estimated by SDS/PAGE, 30% of the variant (3–189) remained so luble under these c onditions. It was purified from the soluble fraction of the cell homogen- ate in the absence o f dithiothreitol, to promote spontaneous formation of the disulfide bond by auto-oxidation. Chro- matography, fi rst o n an anion exchange r esin, then on hydroxylapatite and finally on a size-exclusion gel, resulted in preparations that were at le ast 9 5% pure, as e stimated by SDS/PAGE under r educing conditions (in the presence of 2-mercaptoethanol; Fig. 2B), and with an overall yield of 45% (Table 1). When the (3–18 9) protein was analyzed by SD S/PAGE under nonreducing conditions, two bands of about equal strength were observed (Fig. 2A). Because the spontaneous formation of the disulfide bond was apparently incomplete, total oxidation was achieved by incubating the protein with an excess of Ellman’s reagent (Nbs 2 [32]), leading to a single, but faste r migrating band (Fig. 2C). SDS micelles decorated with disulfide-bonded proteins have a smaller hydrodynamic volume than those d ecorated with the corresponding dithiol forms [33], and therefore migrate more rapidly. In contrast, during culture at 22 °C of the cells producing the ( 64–240) va riant, most of the protein partitioned into the insoluble fraction of the cell homogenate. Attempts to purify this variant by first solubilizing the pre cipitate in guanidinium chloride in the presence or absence of dithiothreitol, an d then dialyzing a gainst phosphate buffer [5], failed to yield significant amounts of soluble material. Apparently, parallel substitution of residues 6 4 and 240 by cysteines prevents the correct folding of the enzyme. The available IGPS structures [1,7] suggest t hat replacement of the long and hydrophobic side chain of the buried and invariable residue M240 by the short, polar side-chain of cysteine m ay disrupt the abundant hydrophobic i nteractions at the C-terminus and hinder the correct folding of the protein. Stu dies with this variant w ere therefore not pursued further. Crystal structure of the oxidized variant eIGPS(3–189) In order to assess the extent to w hich the ( 3–189) disulfide bond had actually formed in the partially oxidized variant eIGPS(3–189) (see Figure 2A), the crystal structure of this protein was solved by X-ray crystallography to 2.1-A ˚ resolution. Unfortunately, t he 4-A ˚ resolution of the previ- ous crystal structure of the monofu nctional e IGPS [25] obstructs comparison to both the structure of the eIGPS domain of the bifunctional enzyme and the structure of the variant reported here. Hence, throughout this work, the structure of the eIGPS domain from the bifunctional enzyme [1] will be u sed as r eference. The Ca tr ace of partially oxidized eIGPS(3–198) very closely superimposes on that of the e IGPS domain (the overall rmsd for Ca atoms is 0.51 A ˚ ), with residues 45–47 and 206–209 involved in crystal packing showing the largest differences. Figure 3 s hows t he enzyme in a state of partial oxidation, as confirmed by SDS/PAGE from dissolved crystals (Fig. 2 A). Indeed, double conformations can be observed for the side-chain of the C3 residue, with one rotamer as part of the d isulfide bridge to C189, and a second rotamer corresponding to the -CH 2 SH side chain of the reduced form. Overall, the structure does not reveal any substantial differences compared to the wild-type eIGPS in the vicinity of the substitutions. Additional electron density was observed, however, in the active site of the protein. It can be modelled as the CdRP substrate (data not shown), but a detailed description including a comparison to related Fig. 2. Partial oxidative closure of the engineered (3–189) disulfide bond. SD S PAGE in a bsence of mercaptoethanol. Lane A, purified and spontaneously oxidized, variant (3–189); l ane B, as in (A), but with dithiothreitol in the sample buffer; lane C, as in (A), but with Nbs 2 ; lanes M, marker proteins with the given M r -values (kDa). Fig. 3. 2F obs -F calc /a calc electron density map showing the disulfide bond contoured at 1.0 r. The newly introduced cysteine residues C3 and C189 are l abelled (black dots, Sulfur atoms). Ó FEBS 2002 Stabilization of a (ba) 8 -barrel protein (Eur. J. Biochem. 269) 1149 complex structures will be reported elsewhere. Perhaps this unexpected feature is responsible for the observed incom- plete autoxidation of eIGPS(3–189) shown in Fig. 2A. The following measurements were conducted with the completely oxidized form (Fig. 2 C). Conformational analysis in solution In order to further analyze possible structural differences between the reduced and o xidized f orms of eIGPS(3–189), designated red(3–189) and ox(3–189), a nd the wild-type eIGPS, we measured far-UV CD spectra in phosphate buffer (Fig. 4). All forms displayed identical spectra within error limits. Therefore, it can be concluded that secondary structural elements are not perturbed by the double replacement in both the d ithiol a nd disulfide forms. Near- UV CD measurements were performed t o further analyze the tertiary structure of eIGPS and its variants. The spectra (Fig. 5) r evealed that eIGPS, red(3 –189) and ox(3–189) have the same minima and maxima, indicating that they have a similar chiral environment for W 15, which is the only tryptophan of the eIGPS domain, and partially accessible to solvent (ASA ¼ 48 A ˚ 2 ). Furthermore, fluorescence mea- surements in phosphate buffer, excited at 295 nm, were employed to monitor polarity changes in the environment of W15 upon disulfide formation (data not shown). The spectra of eIGPS, re d(3–189) and ox(3–189) were identical, implying that the indole moiety of W15 is similarly exposed to solvent in all three cases. The spectra were also characterized by identical fluorescence emission maxima at 348 nm, supporting the conclusion that the indole moiety of W15 is exposed to solvent. Thus, no significant structural differences local to helix a0 s eem to occur in both red(3–189) and o x(3–189), w ith respect to the wild-type. In summary, our near-UV and far-UV CD as well as fluorescence measurements confirm that n either the introduction of the (3–189) disulfide bridge nor specific experimental c onditions affect the structure of the eIGPS dom ain. Thermostability eIGPS can be reversibly unfolded by GdmCl in both Tris [34] and phosphate [35] buffers. Red(3–189) displays the same properties (data not shown). However, the unfolding of ox(3–189) by GdmCl in the absence of dithiothreitol was irreversible, presumably due to thiol-disulfide scrambling [30]. Therefore, t he relative stability of the three forms could only be estimated by irreversible thermal inac tivation (Fig. 6 ) [14]. The results show that the maximal velocities (V max ¼ k cat · [E 0 ]) of the three forms decay irreve rsibly and exponentially at 50 °C. In contrast to eIGPS, ox(3–189) is stabilized 13-fold, whereas red(3–189) is destabilized fivefold, most likely due to the loss of the salt bridge E167- R189. In other words, ox(3–189) is stabilized 65-fold over the d ithiol form, which is the correct reference for estimating Fig. 4. Far-UV CD spectra at 25 °C. s,parentaleIGPS;h, reduced (dithiol); n, oxidized (disulfide) forms of the variant eIGPS (3–189). Protein concentrations were between 10 and 21 l M ;d ¼ 0.1 cm. Buffer: 0,05 M Na-phosphate, pH 7.5, 1 m M EDTA (in case o f red(3– 189) with 1 m M dithiothreitol). Fig. 5. Near-UV CD spectra a t 25 °C. s,parentaleIGPS;h, r ed uced (dithiol); n, oxidized (disulfide) forms of the variant eIGPS (3–189). Protein concentrations were between 10 and 21 l M ;d ¼ 5 cm. Buffer as in Fig. 4. Fig. 6. The disulfide bond of ox(3–189) stabilizes the fold of eIGPS kinetically. Therm al inactivation at 50 °C i s an irreversible, exponen- tial process. eIGPS, reduced (dithiol) and oxidized (disulfide) variants of eIGPS(3–189) were in cubated at concentrations of 10 l M protein in 0.1 M potassium phosphate pH 7.5, 1 m M EDTA. eIGPS and red(3–189) also c ontained 1 m M dithiothreitol. Enzyme activity was determined in samples drawn at the indicated times and quenched on ice. Half-lives: ox(3–189), 49 min; eIGPS, 3.7 min; red(3–189), 0.75 min . 1150 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the effect of closing this disulfide bridge. These results imply that the engineered disulfide bond crosslinks parts of the structure that p robably separate in t he parental protein before the rate-determining step of i ts irreversible unfolding is attained [15]. Thus, t he d isulfide-linked variant apparently unfolds via a transition state that is different from t o that of the wild-type eIGPS. Variation of the phosphate concen- tration between 5 a nd 100 m M revealed that the kinetic stabilities of eIGPS and its variants increase with i ncreasing phosphate concentration (data not shown). These observa- tions support t he idea that phosphate may serve as an additionally stabilizing electrostatic clamp within the active site. Note that phosphate specifically interacts with K55 (loop b1a1), G216 ( loo p b7a7) (not shown i n Fig. 1) a nd the helix dipole of helix a8¢ [1], i.e. protein segments that are far apart in the protein sequence but adjacent in s pace. These considerations could also explain why the second disulfide variant (64–240) does not fold properly, even in the presence of phosphate. M240 is an invariant, solvent- inaccessible residue (ASA ¼ 0A ˚ 2 ) that anchors t he short helix a8¢ to the core of th e protein [1]. Merz et al. [36] have shown that disrup tive substitutions near the closure between the ba1andba8 modules of the (ba) 8 -barrel of sIGPS are generally destabilizing. Replacing M240 of eIGPS with cysteine may therefore destabilize the hydro- phobic interface between the terminal modules ba1and ba8, as well as helix a8¢, t hus decreasing the protein’s affinity for ph osphate. Reactivity of cysteines as a measure for protein flexibility The accessibilities of the three buried cysteine s (C54, C113 and C134) of eIGPS and the dithiol and disulfide forms of the (3–189) variant can be assessed by measuring the kinetics of their irreversible reaction with Nbs 2 [32] [37]. ESH þ DTNB À! k ESTNB þ TNB ð1Þ This reaction, which can be followed by the increase of absorption of TNB at 440 nm, becomes pseudo-first order, i.e. exponential, when Nbs 2 is in excess: Àd½ESH dt ¼ d½TNB dt ¼ k obs ½ESð2Þ where k obs ¼ k[DTNB] is the observed first-order rate constant. Both the total number of reactive cysteine sulfhydryls as well as their average rate constant k as expressed b y t he observed half-lives t 1=2 ¼ ln 2 k½DTNB are presented in Table 2. The measurements were performed in 50 m M phosphate buffer, when the active site is 97% saturated with phosphate [38]. As determined by SDS/PAGE under nonreducing condi- tions (as described in Fig. 2), no i ntermolecular bridges were formed during the oxidation process, as there was no evidence for e ither aggregation o r cross-linking. The possi- bility t hat a further i ntramole cular disulfide bridge had formed between C113 and C134 (cf. Fig. 1) was also excluded b y measurements of the forms in 0.5% SDS, which unfolds the proteins immediately and allows Nbs 2 to react with all free thiols that were not accessible in the folded state. No decrease of the maximally expected numb er o f free sulfhydryl groups was found. Only one of three native cysteines of eIGPS reacted with Nbs 2 , a lbeit slowly. A s the three cysteines are basically solvent inac cessible, as judge d from the calculated a ccessible surface areas (ASA values are: C45 ¼ 1A ˚ 2 ;C113¼2A ˚ 2 ; C134 ¼ 0A ˚ 2 ), we cannot identify the reactive cysteine of eIGPS from structural considerations. In ox(3–189), how- ever, in which the two newly introduced cysteines form a disulfide bond, the reactive cysteine is almost completely protected. In contrast, at least two cysteines of red(3–189) react rapidly. As the positions of T3 and R189 of eIGPS are partially accessible to solvent (ASA values are: T3 ¼ 54 A ˚ 2 ; R189 ¼ 68 A ˚ 2 ), it is likely that C3 and C189 of red(3–189) are the two reactive cysteines. They are converted to the corresponding disulfide via a mixed disulfide intermediate [E(SH)STNB], which does not accumulate. EðSHÞ 2 þ DTNB ! EðSHÞSTNB þ TNB ð3Þ EðSHÞSTNB ! ES 2 þ TNB ð4Þ The cysteine group in excess of C 3 and C189 that reacts to 40% c ompletion in r ed(3–189) is likely the same as that which reacts in eIGPS to 90%. The particularly slow reaction of this parental cysteine in ox(3–198) to only 10% completion must be due to the decreased structural fluctuations of this form of the variant hindering the access of DTNB by comparison to eIGPS. Catalytic constants As the active s ite is l ocated in a d epression at the C-terminal end of the b-barrel, between the structured segments that carry the newly introduced pairs of cysteines (see Fig. 1), enzyme activity is a sensitive monitor for detecting changes in both the structure and flexibility of the three enzymes. Steady-state kinetic measurements were conducted in Tris Table 2. Reaction of protein sulfhydryl groups with Nbs 2 . The protecting effect of th e introduced disulfide bridge. Variants Free sulfhydryl groups per protein chain a Total b Accessible Protected c t d 1=2 (min) eIGPS 3 0.9 2 10 ox(3–189) 3 0.1 3 > 100 red(3–189) 5 2.4 3 < 1 a Buffer: 0.05 M potassium phosphate buffer, pH 7.5, 1 m M EDTA; T ¼ 25 °C. b Evaluated by conducting the reaction at 25 °C in 0.05 M Tris buffer, pH 7.5, 0.5% SDS. c Rounded, integral numbers. d Half-life evaluated from exponential progress curves recorded at 440 nm. Nbs 2 concentration ¼ 1m M . ÓFEBS 2002 Stabilization of a (ba) 8 -barrel protein (Eur. J. Biochem. 269) 1151 buffer and i n the absence of dith iothreitol. Measurements in phosphate buffer are not feasible because phosphate is a competitive inhibitor (K i ¼ 2.8 m M [38]). The Michaelis constants (K CdRP M ) of the two forms of both ox(3–189) and red(3–189) are only % 15% smaller than that of eIGPS (Table 3). The turnover numbers, however, are decreased t o 45% in red(3–189) and to 10% in ox(3–189). A s t he poor activity of the thermostable IGPS from S. solfataric us at low temperature is due to the rate-limiting release of the product IGP [36], it i s likely that ox(3–189) is ‘constipated’ [36] by the rigidified structure. This finding suggests that covalent crosslinking the helix a0 to the loop b6a6is responsible for the retarded release of product in ox(3–189), and i s supported by the decreased reactivity with Nbs 2 of the single most reactive cysteine in o x(3–189), in contrast to eIGPS (Table 2). CONCLUSION We have demonstrated that a mesophilic (b/a) 8 -barrel enzyme from the tryptophan biosynthesis pathway, namely indoleglycerol phosphate synthase from E. coli,canbe stabilized against irreversible thermal denaturation by the introduction of a new disulfide bridge. The new disulfide crosslink of eIGPS(3–189) fastens the N-terminal extension to the catalytic face of the (ba) 8 -barrel fold, thus rigidifying it and changing the pathway of unfolding. Despite obeying the structural criteria of good disulfide geo metry, as well as sufficient distance from both catalytic residues and parental cysteines, the variant eIGPS(64–240) failed to fold to the native structure, even in the r educed state. We conclude that another important criterion is to avoid replacing solvent- inaccessible, hydrophobic residues by cysteines. Although surface disulfide bridges have not generally been selected during the evolution of thermophilic proteins [39], p erhaps because of the chemically reducing e nvironment of the cytoplasm, correctly d esigned disulfide bridges, which form only after release from the cytoplasm, are o f specific interes t for biotechnological applications. ACKNOWLEDGEMENTS The authors thank Drs Thorsten Kno ¨ chel and Ralf Thoma for advice, Dr Reinhard Sterner for critical discussion and Gu ¨ nter Pappen berger for designing the stereo figure. This work was supported by grant Nr. 31–45855.95 of the Swiss National Science Foundation (to K. K.). REFERENCES 1. Wilmanns, M., Priestle, J.P., Niermann, T. & Jansonius, J.N. (1992) Three-dimensional structure of the bifunc tional e nzyme phosphoribosyl anthranilate isomerase: indoleglycerol phosphate synthase from Escherichia coli r efined at 2.0 A ˚ resolution. J. Mol. Biol. 223, 477–507. 2. Eberhard, M., T sai-Pflugfelder, M., Bolewska, K., Hommel, U . & Kirschner, K . ( 1995) Indoleg lycerol p hosphate syntha se- phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains. Biochemistry 34, 5419–5428. 3. Merz, A., Kno ¨ chel, T., Jansonius, J.N. & Kirschner, K. (1999) The hyperthermostable Indolglycerol Phosphate Synthase from Thermotoga maritima is destabilized by mutational dis- ruption o f two solvent-exposed salt bridges. J. Mol. Biol. 288, 753–763. 4. Stehlin,C.,Dahm,A.&Kirschner,K.(1997)Deletionmuta- genesis as a test of evolutionary relatedness of indoleglycerol phosphate syn thase w ith ot her T IM barrel enzymes. FEBS Lett. 403, 268–272. 5. Darimont, B., Stehlin, C., Szadkowski, H. & Kirschner, K. (1998) Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli. Protein Sci. 7, 1221– 1232. 6. Altamirano, M .M., Blackburn, J.M., Aguayo, C. & Fersht, A.R. (2000) Directed evolution of new catalytic activity u sing the a l- pha/beta- barrel scaffold. Nature 403, 617–622. 7.Hennig,M.,Darimont,B.,Sterner,R.,Kirschner,K.& Jansonius, N.J. (1995) 2 .0 A ˚ structure of i ndole-3-glycerol phosphate synthase from the hyperthermophile Sulfolobus sol- fataricus: possible determinants of protein stability. Structure 3, 1295–1306. 8. Sun, D.P., Sauer, U., Nicholson, H. & Matthews, B.W. (1991) Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis. Biochemistry 30, 7142–7153. 9. Betz, S.F. (1993) Disulfide bonds and t he stability o f globular proteins. Protein Sci. 2, 1551–1558. 10. Shaw, A. & Bott, R . (1996) Engineering enzymes for stability. Curr. Opin. Struct. Biol. 6, 546–550. 11. Mansfeld, J., Vriend, G., Dijkstra, B.W., Veltman, O.R., Burg, B.V.D., Venema, G., Ulbrich-Hofmann, R. & Eijsink, V.G.H. (1997) Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond. J. Biol. Che m. 272, 11152–11156. 12. Wakarchuk, W.W., Sung, W.L., Campb ell, R.L., Cunningham, A., Watson, D.C. & Yaguchi, M. (1994) Thermo stabilisation of the Bacillus circulans xylanase by the introduction o f d isulfi de bonds. Protein Eng. 7, 1379–1386. 13. Eder, J. & Wilmanns, M. (1992) Protein engineering of a disulfide bondinab/a-barrel protein. Biochemistry 31, 4437–4444. 14. PlazadelPino,I.,Ibarra-Molera,B.&Sanchez-Ruiz,J.(2000) Lower kinetic limit to protein thermal stability: a proposal regarding protein stability in vivo and its r elation with misfolding diseases. Proteins 40, 58–70. 15. Clarke, J. & Fersht, A.R. (1993) Engineered disulfide bonds as probes of the folding pathway of barnase: i ncreasing the stability of proteins against the rate of denaturation. Biochemistry 32, 4322–4329. 16. Sambrook, J., Fritsch, E.E. & Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 17. Studier, F .W., R o senberg, A .H., Du nn, J.J. & D ubendo rff, J .W. (1990) Use of T7 R NA polymerase to direct e xpression of c loned genes. Methods Enzymol. 185, 60–89. 18. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. & Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68. 19. Sarkar, G. & Sommer, S.S. (1990) The ÔmegaprimerÕ method of site-directed mutagenesis. Biotechniques 8, 404–407. 20. Kirschner, K., Szadkowski, H., Jardetzky, T.S. & Hager, V. (1987) Phosphoribosyl anthranilate isomerase-indoleglycerol- Table 3. Steady-state kinetic constants o f e IGPS, red(3–189) a nd o x (3–189) a . Variant k cat (s )1 ) K CdRP M k cat /K CdRP M (l M )1 Æs )1 )(l M ) eIGPS 2.7 0.30 9.0 ox(3–189) 0.3 0.28 1.1 red(3–189) 1.2 0.24 5.0 a Buffer: 50 m M Tris buffer, pH 7.5, 1 m M EDTA, 25 °C. 1152 A. Ivens et al. (Eur. J. Biochem. 269) Ó FEBS 2002 phosphate synthase from Escherichia coli, Methods Enzymol. 142, 386–397. 21. Cornish-Bowden, A. (1995) Fundamentals of Enzyme Kinetics. Portland Press Ltd, London. 22. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head o f b acteriophage T 4. Na ture 227, 680–685. 23. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of prot ein-dye binding. Anal. Biochem. 72, 248–254. 24. Ellmann, G.E. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 7 0–77. 25. Wilmanns, M., Schlagenhauf, E., Fol, B. & Jansonius, J.N. (1990) Crystallization a nd structure solution at 4 A ˚ resolution of the r ecombinant synthase domain o f N-(5¢-phosphoribosyl)- anthranilate isomerase: indole-3-glycerol-phosphate synthase from Escherichia c oli complexed t o a substrate a nalogu e. Pr otein Eng. 3, 173–180. 26. Otwinowski, Z. & Minor, W. (1997) Processing of X-ray dif- fraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. 27. Navaza, J. (1994) An automated package for molecular replacemen t. Acta Crystallogr. A50, 157–163. 28. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W .L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J S., Kuszewski, J., Nilges, N., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. & War- ren, G.L. (1998) Crystallography and NMR system (CNS): a new software s ystem f or m acromolecula r structu re det ermina- tion. Acta Crystallogr. D54, 905–921. 28a. Collaborative Computational Project, Number 4 (1994) The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D50, 760–763. 29. Swodhamini, R., S rinivasan, N., Shoichet, B., Santi, D.V., Ramakrishnan, C. & Balaram, P. ( 1989) Stereochemical model- ing o f disulfide bridges. Criteria for introduction i nto proteins by site-directed mutagenesis. Protein Eng. 3, 9 5–103. 30. Perry, L.J. & Wetzel, R. (1986) Unpa ired cysteine-54 interferes with t he ability of an e ngineered disulfide to stabilize T4 lyso- zyme. Biochemistry 25, 733–739. 31. Kopetzki, E., Schumacher, G. & Buckel, P. (1989) Con trol of formation of active soluble or inactive insoluble baker’s yeast alpha-glucosidase P I in Escherichia coli by induction a nd gr owth conditions. Mol. General Genet. 216, 149–155. 32. Riddles, P.W., Blakeley, R.L. & Zerner, B. ( 1983) Reassessment of Ellman’s reagent. Methods Enzymol. 91, 4 9–59. 33. Pollitt, S. & Zalkin, H. (1983) Role of primary structure and disulfide bond formation in beta-lactamase secretion. J. Bacte- riol. 153, 27–32. 34. Eberhard, M. ( 1990) Structural and fu nctional studies on engi- neered, monofunctional domains of an originally bifunctional enzyme of Escherich ia coli. PhD Thesis, University of Basel. 35. Darimont, B. (1994) Studies o n catalysis, folding and evolution of indoleglycerol phosphate synthase, an eightfold (ba)-barrel protein involved in tryptophan b iosynthesis, PhD Thesis, Uni- versity Basel. 36. Merz, A., Yee, M.C., Szadkowski, H., Pappenberger, G., Crameri, A., Stemmer, W.P., Yanofsky, C. & Kirschner, K. (2000) Improving the catalytic a ctivity o f a thermophilic enzyme at low temperatures. Biochemistry 39, 880–889. 37. Ballery, N., Desmadril, M., Minard, P. & Yon, J.M. (1993) Characterization o f an intermediate i n th e folding pathway of phosphoglycerate kinase: chemical reactivity of genetically introduced cysteinyl residues during the folding process. Biochemistry 32 , 708–714. 38. Bisswanger, H., Kirschner, K., Cohn, W., Hager, V. & H ansson, E. (1979) N-(5-Phosphoribosyl) anthranilate isomerase-indole- glycerol-phosphate synt hase. 1 . A su bstrate analogue binds to two different binding sites on the bifunctional enzyme from Escherichia coli. Biochemistry 18, 5946–5953. 39. Sterner, R. & Liebl, W. (2001) Thermophilic adaptation of proteins. Crit. Rev. Biochem. Mol. Biol. 36, 39–106. Ó FEBS 2002 Stabilization of a (ba) 8 -barrel protein (Eur. J. Biochem. 269) 1153 . Stabilization of a (ba) 8 -barrel protein by an engineered disulfide bridge Andreas Ivens 1 , Olga Mayans 2 , Halina Szadkowski 3 , Catharina Ju¨. than ePRAI. The catalytic activity of eIGPS decays at 55 °C w ith a half-life o f 0 .5 min [3]. In contrast, ePRAI activity decays at 60 °C with a half-life