Tài liệu Báo cáo Y học: Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species doc

Eur J Biochem 269, 5536–5546 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03259.x Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ Johanna Arnorsdottir1,2, Runa B Smaradottir1, Olafur Th Magnusson2, Sigrıdur H Thorbjarnardottir1, ´ ´ Gudmundur Eggertsson1 and Magnus M Kristjansson2 Institute of Biology, University of Iceland; and 2Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, Iceland The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp PA44 has been successfully cloned, sequenced and expressed in Escherichia coli The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index These factors may contribute to the adaptation of these proteinases to different temperature conditions Many microorganisms and ectothermic animals live under environmental temperatures that fluctuate in the range )2 to 10 °C without the opportunity to regulate their cellular temperatures [1–3] In fact, cold temperature is the most widespread physiological stress condition that organisms have either to adapt to or to avoid Adaptive changes in protein structure and function induced by cold are of prime importance for cold acclimation and survival processes [4] A common denominator of evolutionary adaptive changes of proteins appears to be the conservation and optimization of the functional state of the proteins, such that they are in Ôcorresponding statesÕ with respect to functionally important motions, under the different physical conditions to which the proteins have adapted [5] It has been suggested that in order to maintain such Ôcorresponding statesÕ for efficient biological function at low temperatures, cold-adapted proteins must have adopted a higher degree of conformational flexibility [5–11] As such cold-adaptive strategies would require weakening or alteration of some intramolecular interactions, the structural stability of cold-adapted proteins is expected to be diminished in comparison with their counterparts adapted to higher temperatures [6–8,11,12] This has indeed been generally observed for naturally occurring psychrophilic enzymes studied to date Recent studies in which directed evolution was used to induce cold adaptive properties in a mesophilic enzyme and increased thermal stability in a psychrophilic enzyme have, however, indicated that there may not be a strict correlation between increased activity at low temperatures and decreased thermostability [13–15] In recent years, there has been a growing interest in enzymes from psychrophilic microorganisms, both as models in studies on thermal stability and molecular adaptation of proteins, as well as potential candidates for biotechnological applications Several enzymes from psychrophilic bacteria have now been characterized [16–35] and crystal structures of citrate synthase [23], triose-phosphate isomerase [24], a-amylase [27,28], and that of malate dehydrogenase [31] have been published The psychrophilic enzymes characterized so far generally have higher catalytic activities at low temperatures and are less thermostable than their counterparts from mesophiles Comparative studies where available crystal structures, sequences or threedimensional homology models of psychrophilic proteins have been compared with homologous meso- and/or thermophilic proteins have shown that a general set of rules does not seem to exist for cold adaptation of proteins Coldadaptive mechanisms seem to involve weakening of certain ´ Correspondence to M M Kristjansson, Department of Biochemistry, Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland Fax: + 354 5528911, Tel.: + 354 5254800, E-mail: mmk@raunvis.hi.is Abbreviations: AQUI, aqualysin I; GdmSCN, guanidinium thiocyanate; GdmCl, guanidinium chloride; PRK, proteinase K; Suc-AAPF-NH-Np, succinyl-AlaAlaProPhe-p-nitroanilide; VPR, Vibrio-proteinase Enzymes: aqualysin I (EC 3.4.21.-); proteinase K (EC 3.4.21.64); Vibrio-proteinase (EC 3.4.21.-) Note: the sequence reported in this paper has been deposited in the GenBank database (accession number AF521587) (Received 19 June 2002, revised 11 September 2002, accepted 16 September 2002) Keywords: cold adaptation; psychrotrophic; Vibrio-proteinase; proteinase K-like; subtilisin-like proteinase Ó FEBS 2002 Characterization of a cloned psychrotrophic proteinase (Eur J Biochem 269) 5537 critical intramolecular interactions that tend to facilitate increased local or global flexibilities in the protein molecules Fewer intra or intersubunit salt-bridges [17,18,21,23, 28,31,33], reduction in aromatic–aromatic interactions [17,19,31,33], extended surface loops [17,21–23], fewer prolines in such loops [19,21–23,28,33], lower hydrophobicity [18,19,26,34], weaker calcium-binding [17,27,28,34], improved solvent interactions through additional surface charges [17,19,22,23], and increased exposure of nonpolar groups to the solvent [23,28] have all been cited as possible reasons for increased flexibility and/or decreased thermal stability of proteins from psychrophiles In the case of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus, a single amino acid substitution (Ser238 fi Ala) that eliminates two hydrogen-bonds in a loop region of the molecule could, to a large extent, account for the cold-adaptation of the protein with respect to catalytic activity and thermal stability [24] We previously reported on selected enzymatic characteristics of a subtilisin-like proteinase (subtilase) from a psychrotrophic Vibrio species (VPR) [25] The enzyme belongs to the proteinase K family of these enzymes [36], and showed cold-adaptive properties, i.e higher catalytic efficiency and lower thermal stability when compared with the related mesophilic subtilase, proteinase K (PRK) from the fungus Tritirachium album Limber, and aqualysin I (AQUI) from the thermophile Thermus aquaticus YT-1 We have now cloned, sequenced and expressed the gene for VPR in E coli We compare the deduced amino acid sequence of the proteinase to that of related enzymes from the proteinase K family representing different traits in temperature adaptation MATERIALS AND METHODS Materials The wild-type Vibrio-proteinase (VPRwt) was purified from cultures of Vibrio strain PA44 as described previously [25] Proteinase K, succinyl-AlaAlaProPhe-p-nitroanilide (SucAAPF-NH-Np), guanidinium thiocyanate (GdmSCN), and chemicals used for preparation of buffers were from the Sigma Chemical Company (St Louis, MO, USA) Purified aqualysin I was obtained as described previously [37] Bacterial strains and plasmids The strain used for genomic DNA was Vibrio strain PA44 [25,38] The E coli strain used for cloning was TG1 supE, hsdD5, thi, D(lac-proAB), F¢ (traD36, proAB+, lacIq, lac DZM15) [39] Cloning vectors used were pUC18 and 19, M13 vectors mp18 and 19 (New England Biolabs) The vectors pBAD (Invitrogen) and pJOE 3075.3 [40] were used for gene expression in the E coli strains Top10 and JM109, respectively Growth conditions and DNA manipulations Vibrio PA44 was grown as described [25,38] and genomic DNA was prepared using the CTAB/NaCl method as described [41] For cloning of the proteinase gene primers were designed from the sequence of Vibrio alginolyticus [42]: 5¢-GCGGAATTCTACACCCGCTACATGTGGCGTCG CCAT-3¢ and 5¢-CGCGGATCCTGGGGACTAGATC GAATC-GACCAACGTAA-3¢ Underlined are restriction enzyme sites for EcoRI and BamHI, respectively The primers were used to amplify about 600 base pairs from the genomic Vibrio PA44 DNA The PCR product was cloned into M13mp19 and sequenced The sequencing revealed an EcoRI restriction site on the PCR fragment Genomic Vibrio PA44 DNA was digested with several restriction enzymes (EcoRI, BamHI, HindIII, SalI, SacI, PstI) and analyzed on agarose gels Southern hybridization with the PCR product revealed a HindIII fragment of about 3000 basepairs This fragment was cloned in two EcoRI-HindIII pieces into M13mp19 and sequenced on an ABI 373 automated DNA sequencer (Applied Biosystems) DNA sequences and deduced amino acid sequences were analyzed and compared using Sequencer 3.0 (Applied Biosystems) and the DNASIS software (Hitachi Software Engineering) Homology searches were conducted with the BLAST program using the server at National Center for Biotechnology Information (http://www.ncbi nlm.nih.gov/BLAST/) Expression and protein purification The pBAD TOPO TA CloningÒ Kit from Invitrogen was used for expression The gene was amplified with PCR from genomic Vibrio DNA using the primers 5¢-ATGTTAAA GAAAGTATTAAGTTGTTG-TATTGCAGC-3¢ and 5¢-AAAGTTTGCTTGGAGCGTCAAGCC-ACTGTAAG CCG-3¢, cloned into the expression vector pBAD-TOPO, transformed into the E coli Top10 strain and grown on LB agar containing 100 lgỈmL)1 ampicillin An overnight culture in LB-amp was diluted 50-fold and grown at 37 °C to an D600 ¼ 0.7 At this stage CaCl2 was added to the culture to a final concentration of 10 mM, expression was induced with 0.01% L-arabinose and cells were then grown at 22 °C to an D600 ¼ 1.2 for up to 48 h Cells were harvested by centrifugation (8000 g for 15 min) and lysed in 50 mM Tris, pH 8, 10 mM CaCl2 with mgỈmL)1 lysozyme, sonication and repeated freezing and thawing in liquid nitrogen The cell lysate was treated with lgỈmL)1 DNase and lgỈmL)1 RNase at °C overnight and cell debris was removed by centrifugation (20 000 g for 20 min) Proteins in the supernatant were salted out with ammonium sulfate Pellet from 30 to 60% ammonium sulfate saturation was collected and redissolved in 25 mM Tris, pH 8, 10 mM CaCl2 The recombinant Vibrio-proteinase (VPRrt) was then purified to homogeneity as described for the proteinase isolated from Vibrio strain PA44 [25] Analytical procedures The VPRrt was analyzed for purity/homogeneity and molecular mass by SDS/PAGE using 8–25% gradient gels (PhastGel 8–25) and isoelectric focusing on PhastGel 3–9 using the Phast-System (Pharmacia-LKB) Before electrophoresis the proteinase samples were treated with phenylmethylsulfonyl fluoride to a final concentration of mM to prevent autolysis during sample preparations Gels were stained with Coomassie brilliant blue R-250 Protein concentration in solutions of VPR was estimated by absorbance at 280 nm, using molar absorption coefficients; e(280) of 50 350 M)1Ỉcm)1 and 34 295 M)1Ỉcm)1 for the 40.6 Ó FEBS 2002 ´ ´ 5538 J Arnorsdottir et al (Eur J Biochem 269) and 29.7 kDa forms of the protein, respectively, determined as described by Pace et al [43] Mass spectral data was obtained on a Reflex III MALDI-TOF spectrometer (Bruker), operated in a linear mode The matrix used was sinnepic acid (Bruker) in a 50 : 50 acetonitrile/water mixture, containing 0.1% trifluoroacetic acid, using the dried droplet method Kinetic and thermal stability measurements Enzymatic activity of VPR was assayed using SucAAPFNH-Np as a substrate as described previously [25], using a thermoregulated Unicam UV1 spectrometer Kinetic parameters for activity were determined at 25 °C by fitting the rate data measured at substrate concentrations between 0.05 and mM, to the Michaelis–Menten equation by nonlinear regression using the software KCAT (Biometallics, Inc., Princeton, NJ, USA) Thermal stability of VPRwt and VPRrt was determined by measuring the rates of their inactivation at appropriate temperatures between 52 and 68 °C At each temperature enzyme samples, dissolved (5–10 lgỈmL)1) in 25 mM Tris, pH (adjusted for each temperature), containing 100 mM NaCl, mM EDTA and 15 mM CaCl2, were heated and aliquots were withdrawn at intervals and immediately assayed for remaining activity against SucAAPF-NH-Np as described previously [25] Rate constants for thermal inactivation obtained from the first order plots were used to construct Arrhenius plots describing the temperature dependence of these rate constants [ln k vs 1/temperature (K)] T50% values were obtained from the Arrhenius plots as the temperature at which the rate of inactivation corresponded to 50% loss of original enzyme activity after 30 RESULTS Stability of VPR and related meso- and thermophilic proteinases As reported previously the psychrotrophic Vibrio-proteinase differed significantly with respect to thermal stability in comparison to its mesophilic and thermophilic counterparts, proteinase K and aqualysin I, respectively, when estimated from their rates of inactivation at different temperatures [25] Stability measurements of proteinases are complicated by autoproteolytic cleavage that may take place during unfolding The relationship between unfolding and autolysis is usually ill-defined and in most cases it is not clear to what extent global or local unfolding of the protein molecule has to take place to trigger autolysis In order to obtain an estimate of the conformational stability of the three related proteinases of this comparative study, we also determined the denaturation curves of the enzymes inhibited by phenylmethanesulfonyl fluoride, as a function of denaturant concentration Several subtilases have been reported to exhibit high stability towards protein denaturants, such as urea and GdmCl [44] In this study, the powerful denaturant GdmSCN was used in most experiments as neither urea nor GdmCl unfolded aqualysin I at concentrations set by the upper limit of the aqueous solubility of the denaturants Normalized denaturation curves for VPR, PRK and AQUI as a function of GdmSCN concentration at 25 °C and pH 8.0 are shown in Fig In line with the previous results on thermal stability, a significant difference was observed in the conformational stability of the proteinases, following the order of their respective temperatures of adaptation The [GdmSCN]1/2-values obtained from the curves were 0.55 M, 1.5 M and 3.2 M for the VPR, PRK and AQUI, respectively, underlining large differences GdmSCN denaturation experiments For measuring denaturation curves of VPR, PRK and AQUI as a function of GdmSCN concentration, the proteinases were first inhibited by incubation with 10 mM phenylmethylsulfonyl fluoride for at least 15 min, before the samples were applied to a Sephadex G-25 column, equilibrated with 25 mM Tris, pH 8.0, containing 15 mM CaCl2 and mM EDTA The collected protein peaks were diluted with the buffer containing the different concentrations of GdmSCN After incubation for up to 24 h at 25 °C, the degree of unfolding was monitored by measuring the fluorescence intensity using a Spex Fluoromax spectrofluorometer Reversal of inhibition of the proteinases during incubation was minor and would not affect the pretransition baselines in the denaturation curves The excitation wavelengths were 275 nm for VPR and AQUI and 280 nm for PRK, but emissions were monitored at 355 nm for VPR, at 335 nm for PRK and at 320 nm in the case of AQUI Excitation and emission bandwidths were and nm, respectively Cuvettes were thermostatted at 25 °C The denaturation curves were normalized according to Fu ¼ (yƒ ) y)/ (yƒ ) yu), assuming a two-state transition, where yƒ and yu are the extrapolated fluorescence intensities for the folded and unfolded states, respectively, and y is the measured fluorescence at each point in the transition region Fig Normalized denaturation curves for VPR (d), PRK (j) and AQUI (m), all inhibited by phenylmethylsulfonyl fluoride Unfolding was monitored by changes occurring in the fluorescence emission spectra of the enzymes between 320 and 355 nm as a function of GdmSCN concentration dissolved in 25 mM Tris, pH 8.0, 15 mM CaCl2 and mM EDTA at 25 °C Ó FEBS 2002 Characterization of a cloned psychrotrophic proteinase (Eur J Biochem 269) 5539 in their stabilities towards this denaturant From denaturation curves in the presence of the denaturants GdmCl and urea under the same set of conditions [GdmCl]1/2 and [urea]1/2-values for VPR were found to be 1.3 and 4.4 M, respectively For comparison, [GdmCl]1/2 for PRK was 5.3 M (data not shown) Sequence analysis The sequencing of the vpr gene revealed a 1593-bp sequence encoding a protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa (Fig 2) The deduced sequence has high sequence identity to enzymes of the proteinase K family of subtilisin-like serine proteinases (Fig 3), confirming its former classification as a member of that family [25] As for other enzymes belonging to the proteinase K family [42,45], the VPR sequence consists of three parts; an N-terminal prosequence, a Fig Nucleotide sequence and deduced amino acid sequence of the vpr gene coding for the subtilisin-like proteinase precursor from Vibrio strain PA44 The N- and C-terminal residues of the proteinase domain are underlined and residues of the catalytic triad are enlarged in bold letters proteinase or catalytic domain, and a C-terminal prosequence The N-terminal prosequence of VPR consists of 139 residues and most probably functions as a molecular chaperone for correct folding, but that is subsequently cleaved off by autolysis to give the active proteinase [46–50] Thus, VPR is isolated from cultures of Vibrio strain PA44 as a 40.6-kDa protein, without the 139 residue N-terminal sequence The enzyme undergoes further autolysis under relatively mild conditions where a 100-residue C-terminal extension is cleaved off to give a 29.7-kDa proteinase that remains fully active [25] Both the 40.6-kDa and the 29.7-kDa forms of the enzyme have the same N-terminal sequence, based on amino acid sequencing and MALDITOF mass spectrometry of the two enzyme forms indicates that the peptide bond cleaved between the catalytic domain and the C-terminal extension is at Asp291-Gly292 Thus, according to these results the larger active form of the proteinase is 391 residues with a calculated molecular mass of 40 568 Da and the smaller form is 291 amino acid residues with a molecular mass of 29 670 Da, in good agreement with results obtained by mass spectrometry Comparison of sequences of the proteinase domains of VPR and related enzymes reveals 86 and 71% identity to the mesophilic proteinases from V alginolyticus and V cholerae, respectively, and 60% identity to aqualysin I and the Thermus Rt41A proteinase, but 41% to proteinase K The sequences around the catalytic triad residues (Asp37, His70 and Ser220) are highly conserved in all these enzymes, and except for PRK they apparently all contain identical disulfide bonds, i.e Cys67–Cys99 and Cys163– Cys194 (numbering of VPR from the N-terminal of the proteinase domain (see Fig or 3) The proteinase domain of VPR additionally contains Cys277 and Cys281, and Cys351 and Cys362 in the C-terminal domain, that supposedly also form disulfide bonds as the enzyme has not been found to contain any free thiol groups [25] Identical cysteine residue pattern is found in the V alginolyticus proteinase [42], but of these only the C-terminal disulfide would be present in the enzyme from V cholerae according to its deduced amino acid sequence [51] (Fig 3) The overall amino acid composition of VPR is similar to that of related enzymes, especially to the mesophilic enzymes from V alginolyticus and V cholerae (Table 1) These enzymes all contain relatively high content of Ser and Thr, as well as Gly, probably reflecting a close packing of the polypeptide chain within these protein structures When compared with the thermophilic counterparts, there apparently is a trend to higher content of Asn and Gln in the coldadapted enzyme and also is the number of acidic amino acid residues, especially Asp, higher in the three Vibrio proteinases (Table 1) The thermophilic enzymes, however have higher content of Arg, and relatively higher numbers of hydrophobic amino acids, as well as a higher aliphatic index than the psychro- or mesophilic enzymes (Table 2) When each of the amino acid exchanges that occur between VPR and the other related enzymes were studied, a trend to an increased number of Ser in the cold-adapted enzyme was apparent Especially was Ala fi Ser a frequent thermophilic-to-psychrophilic exchange Eight such Ala to Ser exchanges occur between the structures of AQUI and VPR and seven between the Thermus Rt41 proteinase and VPR Six of the exchanges that occur between VPR and AQUI are also present, however, in the mesophilic enzyme ´ ´ 5540 J Arnorsdottir et al (Eur J Biochem 269) Ó FEBS 2002 Fig Alignment of the deduced amino acid sequence of the psychrotrophic Vibrio-proteinase (VPR), proteinase from Vibrio alginolyticus, aqualysin I (AQUI) from Thermus aquaticus YT-1 and proteinase K (PRK) from Tritirachium album Arrows indicate the N-terminal of VPR as determined by amino acid sequence analysis [25] and the beginning of the C-terminal extension based on results from MALDI-TOF mass spectrometry Residues of the catalytic triad are indicated by an asterisk Secondary structural elements based on known crystal structures of PRK are indicated by h (helix) and s (strand) and calcium binding ligands (P175, V177 and D200 at the Ca1 site and T16 and D260 at the Ca2 site, according to the numbering of the PRK sequence) are denoted by C from V alginolyticus, but all together there are four Ala fi Ser exchanges between VPR and the mesophilic enzyme Comparison of the sequences of the two mesophilic proteinases from V alginolyticus and V cholerae (69% sequence identity) and between the thermophilic AQUI and Thermus Rt41 proteinase (71% sequence identity) indicated that Ala fi Ser amino acid exchanges were not more frequent than other exchanges between these enzymes adapted to similar temperature conditions Fewer proline residues in surface loops has been cited as a possible reason for increased flexibility and or decreased stability in a few psychrophilic enzymes [19,21–23,28,32] Comparison of sequences, as well as 3D homology models of AQUI and VPR, generated by the automatic protein modeling server SWISS-MODEL [52,53], based on known crystal structures of proteinase K, revealed that the thermophilic enzyme contains five Pro residues that are not present in VPR, four (Pro5, Pro7, Pro240, Pro268) of which are located in surface loops It awaits mutagenic studies to test if these proline residues contribute to temperature adaptation of AQUI or VPR Subtilisin-like proteinases are highly dependent on calcium for stability of their native conformations against denaturation and/or autolysis Stronger calcium binding has been suggested as one of the major causes for the high thermal stability of the thermophilic subtilases; thermitase [54,55] and aqualysin I [56] Weaker calcium binding as compared with that of mesophilic counterparts, has also been suggested to be a structural determinant in cold-adaptation of subtilisin S41, from the Antarctic bacterium Bacillus TA41 Ó FEBS 2002 Characterization of a cloned psychrotrophic proteinase (Eur J Biochem 269) 5541 Table Amino acid composition of the proteinase domain of Vibrio-proteinase and related enzymes of the proteinase K family Vibrioproteinase V cholerae Thermus Rt41a AQUI % No % No % No % No % 11.5 2.8 7.0 7.0 2.1 3.1 1.0 12.9 1.4 2.8 5.2 1.7 1.0 2.1 2.4 13.3 6.6 1.4 3.1 11.2 28 11 26 21 13 35 13 15 13 26 22 22 287 9.8 3.8 9.1 7.3 1.4 4.5 1.7 12.2 1.7 4.5 5.2 3.1 1.0 1.7 4.5 9.1 7.7 1.0 2.8 7.7 36 10 14 13 31 11 19 13 22 34 15 24 278 12.9 3.6 5.0 4.7 1.4 2.9 0.4 11.2 2.5 4.0 6.8 1.4 1.1 1.8 4.7 7.9 12.2 1.4 5.4 8.63 40 15 19 13 37 19 2 11 29 25 12 25 281 14.2 5.3 6.8 4.6 1.4 1.8 1.1 13.2 1.8 3.2 6.8 0.7 0.7 1.1 3.9 10.3 8.9 1.1 4.3 8.9 33 12 17 13 33 11 14 37 22 17 19 288 11.8 4.3 6.1 4.7 1.8 2.5 1.8 11.8 1.4 3.9 5.0 2.9 1.8 2.2 3.2 13.3 7.9 0.7 6.1 6.8 V alginol Amino acid No % No Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val S 25 23 21 10 40 18 12 38 20 24 291 8.6 3.1 7.9 7.2 2.1 3.4 1.7 13.7 1.4 3.1 6.2 1.7 1.0 2.4 4.1 13.1 6.9 1.4 2.8 8.2 33 20 20 15 38 19 32 286 PRK Table Comparison of structural parameters based on amino acid composition of Vibrio-proteinase and related enzymes from the proteinase K family Parameter Vibrio-proteinase V alginol V cholerae Thermus Rt41a AQUAI PRK % chargeda % acidicb % basicc Polar/unchargedd Arg/(Arg + Lys) % hydrophobice % aromatic (Ile + Leu)/(Ile + Leu + Val) pIcalcf GRAVYg Aliphatic indexf 15.12 8.93 4.81 49.83 0.643 35.05 6.53 0.529 4.52 )0.268 68.69 13.99 8.04 4.55 48.26 0.615 37.77 6.65 0.418 4.56 )0.038 75.35 17.77 9.06 6.97 46.69 0.555 35.54 5.57 0.560 5.28 )0.433 70.03 12.60 5.04 5.04 46.05 0.882 41.37 8.64 0.555 7.22 0.037 80.07 13.53 5.70 6.05 46.62 0.714 39.85 6.41 0.528 7.89 )0.038 78.90 15.05 6.45 7.17 49.46 0.600 35.49 8.96 0.568 8.25 )0.218 66.52 a DEHKR; b DE; c KR; d GSTNQYC; average of hydropathicity e LMIVWPAF; f Calculated at http://www.expasy.ch/tools/protparam.html; [17] We have also observed a strong dependence on calcium binding for the stability of VPR, as well as for PRK and AQUI (M M Kristjansson, unpublished results) Two calcium sites with different binding affinities have been identified in the high resolution crystal structures of PRK [57,58] At the stronger Ca1-site, calcium ion is coordinated in the form of pentagonal bipyramid by Od1 and Od2 of Asp200, the carbonyl oxygens of Pro175 and Val177, and four water molecules At the weaker Ca2-site, which bridges two loops of the C- and N-termini of the protein, calcium is coordinated by the carboxylate oxygens of Asp260 and the backbone oxygen of Thr16, in addition to 4–5 water molecules [57,58] Comparison of the sequences and the homology models for VPR and AQUI to that of PRK g GRAVY, grand indicate that the Ca1 site appears to be present in both VPR and AQUI The weaker Ca2 site of PRK does not appear to be present, however, in either of the two proteinases, due to the lack of a carboxylate group at positions corresponding to Asp260 in PRK A weakly bound calcium ion has been shown to have a significant role in the thermal stability of AQUI [56], thus, a yet-unidentified Ca-site is likely to be present in that molecule Expression of the vpr gene in E coli Attempts to design a suitable expression system for the vpr gene involved three expression vectors We did not manage to clone the gene into the pET 23b vector, Ó FEBS 2002 ´ ´ 5542 J Arnorsdottir et al (Eur J Biochem 269) possibly because of detrimental background expression The gene was cloned into another vector, pJOE 3075.3 [40] The gene was overexpressed using the pJOE vector in E coli strain JM109, but the protein formed inclusion bodies Changing the conditions in the expression culture, such as lowering the temperature or changing the concentration of the inducer has not given production of active enzyme, nor have attempts to refold the protein by different means in vitro been successful The gene was moderately expressed, giving a yield of  mgỈL)1, using the pBAD TOPO expression system No activity was detected after induction and culturing at 37 °C Culturing at 32 °C after induction resulted in a detectable production of active proteinase, which was further enhanced by lowering the growth temperature to room temperature after induction Characterization of the recombinant VPR The purified VPRrt showed identical properties to VPRwt with respect to migration on SDS/PAGE and the time dependent autoproteolytic 40.6 fi 29.7 kDa conversion at 40 °C (Fig 4) The recombinant and wild-type enzyme also showed identical electrophoretic behavior for both forms of the enzyme on IEF gels (data not shown) The larger form having an estimated isoelectric point of 4.6, but 3.7 for the smaller form A feature of the Vibrio-proteinase is its sensitivity to treatment with dithiothreitol In the presence of 10 mM dithiothreitol at 25 °C, the activity of enzyme is decreased to about one-quarter of its original activity, an effect that was attributed to an eightfold increase in Km for SucAAPF-NH-Np, while the kcat was unchanged for the reaction [25] When VPRrt was treated with dithiothreitol under the same conditions as VPRwt both enzymes displayed identical inactivation behaviour, indicating that the same disulfide(s) are involved in maintaining the integrity of active protein structure in both wild-type and recombinant VPR The wild-type and recombinant forms of the enzyme showed no significant differences in terms of thermal stability when compared in the form of Arrhenius plots (Table 3) They also had comparable Michaelis– Menten kinetic parameters when their amidase activity against succinyl-AAPF-NH-Np was measured at 25 °C (Table 3) Table Comparison of thermal stability (T50%) and Michaelis–Menten parameters (Km and kcat) against succinyl-AAPF-p-nitroanilide of wildtype (VPRwt) and recombinant (VPRrt) forms of the Vibrio-proteinase Property VPRwt VPRrt T50% Km kcat 56 °C 179 ± 12 lM 68.9 s)1 56 °C 164 ± 17 lM 76.5 s)1 DISCUSSION The extracellular proteinase K-like proteinase from the psychrotolerant Vibrio strain PA44 is produced as a 55.7-kDa precursor protein, containing an 139 residue N-terminal prosequence, a 291 residue proteinase domain, and a 100 residue C-terminal domain Production of such relatively large precursor proteins appears to be a common trait of proteinases belonging to the proteinase K family In aqualysin I, from the thermophile Thermus aquaticus, a 127residue N-terminal prosequence is cleaved off in an intramolecular autocatalytic reaction apparently after assisting in the correct folding of the proteinase [49,50,59] The N-terminal prosequence is necessary for the production of active aqualysin I and most likely acts as an intramolecular chaperone, by similar mechanisms as has been described for such N-prosequences of subtilisins [46–48] and a-lytic protease, a trypsin-like serine proteinase [60] Because of the similarity that exists between the sequences of VPR and AQUI, we predict that the N-terminal prosequence of the cold-adapted enzyme has such an intramolecular chaperonelike activity for that enzyme A 105 residue C-terminal prosequence has been found to play an important role in extracellular secretion of AQUI in an expression system using Thermus thermophilus cells, and it was suggested that the sequence is required for the translocation of the precursor across the outer membrane [61] VPR is secreted both as the wild-type enzyme in cultures of Vibrio strain PA44 and in our E coli expression system as a 40.6-kDa protein, containing the C-terminal prosequence and as we have shown previously [25], the enzyme undergoes, under relatively mild conditions, an autocatalytic cleavage to a 29.7-kDa proteinase, lacking the C-terminal extended sequence, but which remains fully active We observed the same behaviour for the recombinant VPR (Fig 4) With Fig In vitro processing of wild-type (A) and recombinant (B) VPR at 40 °C Samples of the enzymes were incubated in 25 mM Tris, pH 8.0, containing 10 mM CaCl2 Aliquots were withdrawn at intervals and phenylmethylsulfonyl fluoride was added to a final concentration of mM to inhibit enzyme activity, followed by analysis by SDS/PAGE on 8–25% gels Ó FEBS 2002 Characterization of a cloned psychrotrophic proteinase (Eur J Biochem 269) 5543 respect to the enzyme characteristics compared in this study, the properties of wild-type and recombinant forms of VPR were undistinguishable, hence we conclude that the two forms are identical molecular entities The deduced amino acid sequence of VPR shows over 50% identity to sequences of the proteinase K-like proteinases from Vibrio alginolyticus [42], Vibrio cholerae [51], Alteromonas sp [62], Kytococcus sedentarius (SWISSPROT accession no Q9L705), aqualysin I from Thermus aquaticus [45] and Thermus strain Rt41A [63] We chose to compare the sequence of VPR to the enzymes from V alginolyticus and V cholerae, as close homologs of VPR of mesophilic origin, the thermophilic AQUI and Thermus Rt41A proteinase, as well as proteinase K as the representative enzyme of this proteinase family, in an attempt to observe tendencies in amino acid substitutions that might correlate with their differences in temperature adaptation Although such a comparison is limited by the small sample size, there appears to be a tendency towards a higher content of polar/ uncharged amino acids, in particular Asn, Gln and Ser, in the cold-adapted VPR, especially when compared with the thermophilic enzymes Of specific amino acid substitutions, the Ala fi Ser exchange was the most often observed thermophilic-to-psychrophilic substitution It is of interest in this respect that in a recent study where 115 protein sequences from the genome of the extreme thermophilic archaeon Methanococcus jannaschii were compared with their homologs from several mesophilic Methanococcus species, the strongest correlation with thermophily of the observed amino acid exchanges was the decrease in the content of uncharged polar residues (Ser, Thr, Asn and Gln) [64] Of the specific amino acid replacements, the Ser fi Ala exchange showed the highest correlation with thermophily [64] The Ser fi Ala replacement was also observed as one of the most frequent ÔthermostabilizingÕ amino acid exchange observed by Argos and coworkers [65,66] in their sequence comparisons of meso- and thermophilic proteins We are now carrying out mutagenic studies on VPR to examine whether some of the Ala fi Ser substitution we have observed in this comparison may contribute to its cold adaptation The cold-adapted VPR also seems to be less hydrophobic than the thermophilic Thermus proteinases, as calculated in Table The lower hydrophobic content and aliphatic index of VPR can largely be accounted for by the lower Ala content Four Pro residues located on surface loops in AQUI, that are not present in VPR, may contribute to the higher stability of the thermophilic as compared with the cold-adapted enzyme As a result of inherent constraints on rotations around the N–Ca bond of proline residues, their introduction into mobile regions of proteins, such as loops, is expected to restrict available backbone configurations and thus increase rigidity and hence stability in such regions [67,68] Stabilization of loops may also take place by shortening or deletion, or by substitutions that reduce the number of ÔflexibleÕ glycines in such regions [67–69] In addition to restricting flexibility in the loop regions, these factors would also reduce the conformational entropy of the unfolded state of the protein and thus contribute to the stability of the native state [67–69] Conversely, the opposite effects may be important to increase flexibility in cold-adapted enzymes and extended surface loops [17,21–23] and fewer prolines in such loops [19,21–23,28,32], have indeed been reported as structural determinants of cold-adaptation in psychrophilic enzymes Glycine residues are highly conserved in the sequences of the proteinases compared in this study For example, of the 37 glycines present in the proteinase domain of AQUI, 29 are present at identical positions in VPR The catalytic domain of VPR contains 10 additional glycine residues; of those, however, seven are also present at identical sites in the mesophilic proteinase from V alginolyticus According to our homology model only one of the three extra glycines in the cold-adapted proteinase is located in a loop region of the protein Thus an increased number of ÔflexibleÕ glycines in loops is not expected to play a key role in cold-adaptation of VPR As for other subtilisin-like proteinases, calcium binding is highly important for the stability of VPR, but its role in temperature adaptation is far from being straightforward Both site-directed [20] and random mutagenesis (directed evolution) studies [13–15] have shown that modification of groups involved in calcium binding in these proteinases can significantly affect both thermal stability and catalytic activity Sequence comparisons and evaluation of 3D homology models for VPR and AQUI, indicate that at least one of the two well-defined calcium binding sites (the stronger Ca1-site) in the structure of PRK is also present in these enzymes, but the weaker, Ca2-site, cannot be present due to the lack of the carboxylate ligand corresponding to Asp260 in PRK For VPR it is interesting to observe that a highly similar amino acid sequence as the one that makes up one of the three calcium binding sites (the Ômedium strengthÕ Ca2-site) identified in thermitase is present in the enzyme (Fig 5) This Ca-site is positioned in a loop that leads into an a-helix containing the active site His residue of the catalytic triad Except for one substitution (Ala66 fi Ser) the sequences of VPR and the V alginolyticus proteinase are identical in this region, but are different from AQUI In addition to the ligands indicated in Fig 5, an Arg residue (Arg102) stabilizes the Ca-site by making a salt bridge to the ligands Asp57 and Asp60 [54,55] Both of the Vibrio sp enzymes, as well as AQUI and the Thermus Rt41 proteinase, also contain a conserved Arg residue at equivalent sites in their sequences (Fig 3) It still remains to be seen if a difference in calcium binding affinity in any way affects the temperature adaptation among these proteinases In common with many cold-adapted enzymes characterized so far, VPR is anionic, e.g it has an acidic isoelectric point It apparently shares this property with its mesophilic counterparts (Table 3), but is different from the basic pIs for AQUI (pI >9–10) [70] and the Thermus Rt41A proteinase (pI  10.25–10.5) [63] The lower pI of VPR compared with its thermophilic counterparts results from higher content of Fig Comparison of the amino acid sequence of the main ligands (indicated in bold) of the medium strength calcium binding site (Ca2 site) of thermitase and a similar sequence found in VPR The active site His residue is indicated by an asterix ´ ´ 5544 J Arnorsdottir et al (Eur J Biochem 269) Asp and lower number of Arg residues (Table 3) In subtilisin S41, an acidic pI was mainly attributed to 11 extra Asp residues located on the surface of the molecule giving rise to a more flexible enzyme [17] Similar trends have also been observed in a cold active citrate synthase [22] It has been pointed out, however, that subtilisin S41 shares the high Asp content with the closely related subtilisin SSII from the mesophilic Bacillus sphaericus and it might therefore contribute less to cold-adaptation of the psychrophilic enzyme than suggested [13] ACKNOWLEDGMENTS ´ We thank Dr Gudni A Alfredsson, Institute of Biology, University of Iceland for providing us with cultures of Vibrio strain PA44, Dr Hiroshi Matsuzawa, Department of Biotechnology, The University of Tokyo, ´ for samples of aqualysin I, and Dr Olafur H Fridjonsson, Prokaria, Reykjavı´ k for the pJOE 3075.3 vector for these studies We also thank Berit Nielsen at deCODE Genetics, Reykjavik for carrying out the mass spectrometry of samples This work was supported by grants from the Icelandic Research 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Table Comparison of structural parameters based on amino acid composition of Vibrio -proteinase and related enzymes from the proteinase K family Parameter Vibrio -proteinase V alginol V cholerae... activity against succinyl-AAPF-NH-Np was measured at 25 °C (Table 3) Table Comparison of thermal stability (T50%) and Michaelis–Menten parameters (Km and kcat) against succinyl-AAPF-p-nitroanilide of. .. cloning of the proteinase gene primers were designed from the sequence of Vibrio alginolyticus [42]: 5¢-GCGGAATTCTACACCCGCTACATGTGGCGTCG CCAT-3¢ and 5¢-CGCGGATCCTGGGGACTAGATC GAATC-GACCAACGTAA-3¢