Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan
Correspondence
Yasuhiro Takada
ytaka{at}sci.hokudai.ac.jp
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ABSTRACT |
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Present address: Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
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INTRODUCTION |
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Directed modification of the enzyme proteins by random PCR mutagenesis is a useful approach to examine their adaptation mechanism. By comparing the amino acid sequences of mutated enzyme proteins that exhibit thermodynamic properties different from those of the wild-type enzyme, the cause of the alteration can be specified. Such studies for thermophilic (Suzuki et al., 2001; Lönn et al., 2002
), mesophilic (Taguchi et al., 1999
; Wintrode et al., 2000
) and psychrophilic enzymes (Miyazaki et al., 2000
) have been reported. Even among mutated enzymes with similar characteristics to each other, various patterns of amino acid substitution have been observed (Suzuki et al., 2001
). Furthermore, the substituted amino acid residues in a cold-adapted mutant (P3C9) of mesophilic subtilisin SSII were not necessarily identical to the corresponding residues of the naturally cold-adapted subtilisin S41 (Wintrode et al., 2000
), supporting the view that nature has not tested all possibilities for the adaptation of proteins to low temperature (Gerday et al., 1997
).
Cold adaptation of enzymes in central metabolism should be essential for the survival and growth of psychrophiles at low temperatures. Isocitrate lyase (ICL; EC 4.1.3.1) catalyses the cleavage of isocitrate to glyoxylate and succinate, and plays important roles in the metabolism of acetate and fatty acids in micro-organisms and higher plants as a key enzyme of the glyoxylate cycle (Kornberg, 1966; Vanni et al., 1990
; Cozzone, 1998
). We previously reported that the ICLs of two psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea, are homotetrameric, as are their counterparts in other organisms, including Escherichia coli, that they are typical cold-adapted enzymes, and that the expression of the genes encoding these enzymes is cold-inducible (Watanabe et al., 2001
, 2002a
, 2002b
). Furthermore, the recently resolved crystal structures of ICLs and their complexes with ligands from several organisms can provide us with useful information to understand their catalytic function (Britton et al., 2000
, 2001
; Sharma et al., 2000
). We previously found that about 20 amino acid residues among those strictly conserved in ICLs from various organisms are substituted in the corresponding enzyme of C. maris (CmICL) (Watanabe et al., 2002a
). Furthermore, some of the substitutions were found in amino acid residues essential for the catalytic function of E. coli ICL (Ko & McFadden, 1990
; Ko et al., 1991
, 1992
; Diehl & McFadden, 1993
, 1994
; Rehman & McFadden, 1996
, 1997a
, b
, c
) or the adjacent residues. Similar substitutions of conserved amino acid residues have also been observed in other cold-adapted enzymes (Davail et al., 1994
). In this study, to examine whether such substituted residues are related to the cold adaptation of CmICL, they were exchanged for amino acid residues homologous to mesophilic counterparts by site-directed mutagenesis, and the properties of the mutated ICLs were investigated.
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METHODS |
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Site-directed mutagenesis.
Site-directed mutagenesis was carried out by a standard PCR method. The following primers were used to introduce the mutations into CmICL: sense primer207 and antisense primer207 for the substitution of His for Gln207 were 5'-GGTGCTTGTGCTCTTCATATTGAAAACC-3' (28-mer) and 5'-GGTTTTCAATATGAAGAGCACAAGCACC-3' (28-mer), respectively; sense primer217 and antisense primer217 for the substitution of Lys for Gln217 were 5'-GCTGATGAAAAAAAATGTGGACATCAAGACG-3' (31-mer) and 5'-CGTCTTGATGTCCACATTTTTTTTCATCAGC-3' (31-mer), respectively. The mutated regions are underlined. Except for the use of 100 pmol of each primer, the PCR was performed as described above. Both the PCR products were ligated into pTrcHisB to obtain plasmids pHis-Cm207 (the Q207H mutation) and pHis-Cm217 (the Q217K mutation). A plasmid carrying both of the mutations (pHis-Cm207/217) was obtained by PCR with the plasmid pHis-Cm207 and a set of sense and antisense primers217. The mutated plasmids were transformed into E. coli TOP10 and purified. To verify the introduced mutations, the relevant regions of the plasmids were sequenced in both directions, as described above.
Overexpression and purification of His-tagged ICLs.
The E. coli TOP10 cells carrying one of the expression vectors pHis-CmWT, pHis-Cm207, pHis-Cm217, pHis-Cm207/217 or pHis-EcWT were grown at 37 °C in Super broth medium (12 g tryptone, 24 g yeast extract, 5 ml glycerol, 3·81 g KH2PO4 and 12·5 g K2HPO4 per litre, pH 7·0) containing 50 mg ampicillin l1 until OD600 of the culture reached 0·6. The cultures were rapidly cooled on ice and were further incubated for 1824 h at 15 °C after the addition of 0·1 M IPTG. Cells were then collected and resuspended in Buffer A (50 mM sodium phosphate, pH 6·85, containing 2 mM MgCl2, 0·5 M NaCl, 10 mM 2-mercaptoethanol and 10 mM imidazole), employing 20 ml of buffer per litre of the original culture medium. Hen-egg lysozyme (2 mg ml1) was added to the cell suspension, and the mixture was gently shaken overnight at 4 °C. The cells were then disrupted by ultrasonic oscillation. After the centrifugation of the cell lysate at 39 120 g for 30 min at 4 °C to remove cell debris, the supernatant was centrifuged at 65 600 g for 6 h at 4 °C. The resultant supernatant was loaded onto a Ni-NTA column (25 ml; Qiagen) equilibrated with Buffer A. After thorough washing with the same buffer, the column was further washed with 50 ml Buffer B (Buffer A containing 10 %, v/v, glycerol and 20 mM imidazole instead of 10 mM imidazole) and next with 50 ml Buffer C (Buffer B containing 50 mM imidazole instead of 20 mM imidazole). In each case the enzyme was then eluted with 50 ml of Buffer D (Buffer B containing 250 mM imidazole instead of 20 mM imidazole), and the elutant was concentrated with polyethylene glycol #20 000 and dialysed against Buffer E (20 mM potassium phosphate, pH 6·85, containing 2 mM MgCl2, 0·5 M NaCl, 1 mM DTT and 0·01 % NaN3). All His-tagged recombinant ICLs were stored at 4 °C and used in further experiments within a week.
PAGE.
SDS-PAGE was carried out by the method of Laemmli (1970) with a 10 % gel at 25 mV.
Western blot analysis.
After SDS-PAGE of the purified ICLs and the sonicated extracts of the E. coli transformant cells, the proteins on the gels were transferred onto a nitrocellulose membrane (Hybond-C; Amersham Pharmacia Biotech). Western blot analysis was carried out with the ECL Western blotting detection system (Amersham Pharmacia Biotech) and either rabbit antibody against the C. maris ICL (Watanabe et al., 2002a) or mouse antibody against the additional (His)6Gly sequence at the N-terminal of the overexpressed ICLs (Invitrogen). Native ICL of C. maris was purified as described previously (Watanabe et al., 2001
).
Enzyme assay.
Unless otherwise stated, the ICL activity was assayed at pH 6·85 as described previously (Watanabe et al., 2001). Optimum pH for activity was determined with the following buffers: potassium phosphate at pH 6·07·6 and Tris/HCl at pH 7·58·0. Protein was measured by the method of Lowry et al. (1951)
with BSA as a standard.
Measurement of kinetic and thermodynamic parameters.
The kinetic parameters, Km and Vmax, of ICL were calculated by LineweaverBurk analysis. Molecular masses of the wild-type and the mutants of CmICL (Cm-Q207H and Cm-Q217K) and EcICL with the additional N-terminal His-tags were calculated as 246 641, 246 677, 246 641 and 204 124 Da, respectively, from the gene sequences. The activation energies (Ea) of the enzymes were calculated by Arrhenius plotting. The thermodynamic activation parameters were calculated according to the following equations.
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Digestion with trypsin.
The purified recombinant ICLs were dialysed overnight at 4 °C against 0·1 M NaHCO3 (pH 8·1). The ICL enzymes (100 µg) were then digested at 4 °C with 0·1 µg trypsin in the same buffer. At appropriate times after the addition of trypsin into the reaction mixture, the mixture was withdrawn and immediately analysed by SDS-PAGE.
Circular dichroism (CD) spectra of recombinant ICLs.
All purified recombinant ICLs were dialysed overnight at 4 °C against 20 mM potassium phosphate buffer (pH 6·85) containing 2 mM MgCl2 and 1 mM DTT. UV-CD of the ICLs was measured at 20 °C with a J-725 spectropolarimeter (Jasco) by using a cuvette with a 0·1 cm path length. The protein concentrations were determined by the absorbance at 280 nm from the extinction coefficients 53056·5 and 71946·0 M1 cm1 for three CmICLs (wild-type and two mutants) and EcICL, respectively, which were calculated as reported previously (Wetlaufer, 1962). The contents of the secondary structures in the ICLs were estimated with program K2D (Andrade et al., 1993
).
Phylogenetic analysis.
Phylogenetic analysis was done with a CLUSTAL-W program with the unrooted neighbour-joining method. The phylogenetic tree was produced with the program TreeView 1.6.1 distributed by the Bioinformatics Center of Kyoto University, Japan (www.genome.ad.jp/Japanese/).
Molecular modelling of CmICL.
The three-dimensional structural model of CmICL was built on the basis of its homology to EcICL (PDB No. 1IGW) with the program SWISSPDB VIEWER.
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RESULTS |
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Kinetic and thermodynamic parameters of the ICLs
The effects of temperature on the kinetic parameters of recombinant CmICLs and Ec-WT were examined (Fig. 4). Km values for isocitrate (Kmisocitrate) of all ICL enzymes tested were increased by elevating temperature. Whereas the Kmisocitrate values of Cm-Q207H were 2·23·5-fold larger than those of Cm-WT, the values of Cm-Q217K were 0·60·8 times those of Cm-WT, and were almost the same as those of Ec-WT. On the other hand, catalytic efficiencies (kcat/Kmisocitrate) of these ICLs were maximal near the optimum temperature for activity of each enzyme (Fig. 4b
). The catalytic efficiency of Cm-WT at 10 °C was 1·2-fold that of Ec-WT and was 41·3 % of its maximum value at 20 °C, while the relative efficiency values of Cm-Q207H, Cm-Q217K and Ec-WT at the same temperature were only 13·5, 23·3 and 15 %, respectively. These results indicate that the catalytic function of both Cm-Q207H and Cm-Q217K was impaired at low temperature.
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DISCUSSION |
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Two amino acid residues, His184 and Lys194, of EcICL are known to be conserved in many prokaryotic and eukaryotic ICLs, but both of the corresponding residues in CmICL are Gln (Fig. 1a). Lys194 of EcICL should be essential for the catalytic function, because an active-site loop including this residue moves flexibly when the enzyme binds to the substrate and releases the reaction products (Britton et al., 2000
, 2001
; Sharma et al., 2000
; cf. Fig. 7
). Between 30 °C and 40 °C, Cm-Q217K has a specific activity similar to that of Cm-WT (Fig. 3a, b
) and the Kmisocitrate value of the former was lower than that of the latter (Table 1
and Fig. 4a
). These results suggest that, in this temperature range, this mutation strengthens the affinity for isocitrate without diminishing the catalytic function. As reported for EcICL (Rehman & McFadden, 1997b
), the substitution of the cationic Lys residue for Gln should facilitate the binding to the Mg2+isocitrate complex as substrate. On the other hand, the catalytic rates of Cm-Q217K between 10 °C and 25 °C were obviously decreased (Figs 3a and 4b
), indicating that the Gln residue is important for cold adaptation of CmICL. This may be due to a decrease in the structural flexibility of the mutated enzyme, revealed as an increase in thermostability and a change in the CD spectrum (Figs 3c and 6
, Table 1
). The high flexibility enhances the accommodation of enzyme with substrate at low temperature, but is also responsible for poor binding to ligand. It has been reported that the Km values of cold-adapted enzymes are often higher than those of mesophilic and/or thermophilic counterparts (Fields & Somero, 1998
). In fact, between 10 °C and 30 °C, the Kmisocitrate of Cm-WT was larger than those of Ec-WT and Cm-Q217K (Fig. 4a
).
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A decrease of H# (or Ea) is one of the common strategies for psychrophilic enzymes to weaken the temperature dependence of kcat (Lonhienne et al., 2000
). Furthermore, as seen in the comparison of Cm-WT and Ec-WT, the T
S# values of psychrophilic enzymes are also generally known to be smaller than those of their mesophilic counterparts, because the enhanced flexibility of psychrophilic enzyme proteins allows them to diversify the transition states of intermediate enzymesubstrate complexes during the reaction more than mesophilic ones (Fields & Somero, 1998
; Lonhienne et al., 2000
). Conversely, such a relationship was not observed in the comparison of Cm-Q207H and Cm-Q217H with Ec-WT (Table 1
). Therefore, with regard to their thermodynamic parameters also, both the mutated CmICLs are judged to be inferior to Cm-WT as a cold-adapted enzyme, although they still exhibit a lower optimum temperature for activity and higher thermolability than Ec-WT.
The phylogenetic analysis revealed that, among the subfamily 3 ICLs from a limited number of eubacteria including C. maris, Gln207 and Gln217 of CmICL are conserved, while the putative ICLs of other psychrophilic and psychrotrophic bacteria are classified into subfamily 1, separate from CmICL, and possess His and Lys, identical to EcICL, at the corresponding positions (Fig. 1). This implies that the importance of the two Gln residues to cold adaptation is specific to CmICL, and agrees with the finding that more than one pattern of amino acid substitution contributes to the cold and/or thermal adaptation of proteins (Fields & Somero, 1998
; Sheridan et al., 2000
; Suzuki et al., 2001
; Lönn et al., 2002
; Gerike et al., 2001
). On the other hand, the possibility remains that additional amino acid residue(s), interacting and/or cooperating with the two Gln residues, are involved in the cold adaptation of CmICL. Therefore, further study is in progress to confirm this possibility.
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ACKNOWLEDGEMENTS |
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Received 31 March 2004;
revised 29 July 2004;
accepted 30 July 2004.
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