1 Department of Molecular Biology and Cell Biology and 2 The Key Lab of Structural Biology, USTC, CAS, School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026, China
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Abstract |
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Keywords: molecular modelling/thermostability/xylose isomerase
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Introduction |
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This paper describes an experiment to enhance the heat stability of D-xylose isomerase (XI; D-xylose ketol isomerase, EC.5.3.1.5) from Streptomyces diastaticus No. 7, strain M1033 (S.M33 XI) by site-directed mutagenesis. D-Xylose isomerase is usually referred to as glucose isomerase (GI). It can convert xylose into xylulose and catalyze glucose into the sweeter fructose. It has been widely used in the food industry for high fructose corn syrup (HFCS). Since the fructose/glucose equilibrium increases with temperature (Smith et al., 1991), enzymes with stability above 80°C are ideal targets for protein engineering (Smith et al., 1991
). One of the successful examples is that of the K253R mutant of XI from Actinoplanes missouriensis, which increased thermostability 5-fold with respect to wild-type (Mrabet et al., 1992
). However, the same mutant (K253R) of S.M33 XI apparently decreased the heat stability (Wu et al., 1996
).
We have sequenced the S.M33 XI gene (Wang et al., 1994) and expressed it in Escherichia coli (Cui et al., 1993
). The crystal structure of S.M33 XI has been determined at 1.9 Å resolution (PDB code, 1clk; Zhu et al., 1996
). The next step is to improve the thermostability of S.M33 XI by protein engineering. In this work, we have investigated the stability of seven single (Q20L, F53L, N184D, A198C, G138P, G247D and K253R) mutants and one double (G138P/G247D) mutant of S.M33 XI (Zhu et al., 1998
). In both the G138P and G138P/G247D mutants, the thermostability was significantly enhanced. We therefore concluded that thermostability can be improved by introduction of a proline residue into a turn of a random coil.
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Materials and methods |
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Phage M13 mp19 was used for recombinant plasmid construction and sequencing. Escherichia coli strains CJ236 and JM109 were used for site-directed mutagenesis. The pTKD-GI plasmid and E.coli K38/pGP1-2 were used as vector and host for the expression of the cloned XI gene (Cui et al., 1993). All enzymes used for DNA manipulations were from Boehringer and were used according to the manufacturer's specifications. DEAESepharose Fast Flow column and Sephacryl S-300 High Resolution column were from Pharmacia. All other chemicals were of analytical grade for biochemical use.
Site-directed mutagenesis and plasmid construction
Construction of two single mutants was carried out by site-directed mutagenesis according to the double-primer method (Zoller et al., 1987) combined with Kunkel's method (Kunkel et al., 1987
). A 0.9 kb EcoRISphI fragment of the XI gene was extracted and cloned into phage M13 mp19. Single-stranded DNA containing uridines in E.coli CJ236 was extracted as a template and annealed with a synthetic primer with the target substitution. DNA extension and ligation reactions were performed in vitro. Single-stranded DNA carrying the desired mutation was prepared from each single plague, and sequenced in order to check for the correct introduction of the required mutation. The entire XI gene was sequenced to eliminate the possibility of other unexpected mutations. The primers used for mutagenesis were 5'-TACGTCGCCTGGCCCGGCCGCGAGG-3'(G138P) and 5'-ACCTCAACGACCAGTCCG-3'(G247D). Mutations were designed to remove the original restriction sites so as to facilitate the selection of mutants. The 0.9 kb EcoRISphI fragment of pTKD-GI was replaced by the same fragment containing the mutated sites 138 or 247 for expression of the two single mutated genes. The expression plasmid of the double mutant (G138P/G247D) was constructed by the substitution of a 0.7 kb XhoI fragment containing the Asp247 mutated site for that of pTKD-GI (G138P).
Enzyme expression and purification
Escherichia coli K38 containing the wild-type or mutant XI gene was grown in LB medium to an OD600 of 1.5 at 30°C for 57 h and induced at 42°C for 30 min, then expressed at 30°C for another 23 h. Cell pellets were harvested by centrifugation and sonicated to break up the cells. Crude extracts were applied to a DEAESepharose Fast Flow column equilibrated with 50 mM TrisHCl, pH 7.5 containing 10 mM MgCl2, 100 mM NaCl and 0.1 mM phenylmethanesulfonyl fluoride (PMSF). The proteins were partially purified by eluting with a linear NaCl gradient from 0.1 to 0.5 M. Eluate containing XI activity was pooled, concentrated and loaded onto a Sephacryl S-300 High Resolution column which was equilibrated with 50 mM TrisHCl, pH 7.5 containing 10 mM MgCl2, 100 mM NaCl and 0.1 mM PMSF. XI was eluted with 0.1 M NaCl. Enzyme purity of wild-type and mutant forms of XI were evaluated by 12% SDSPAGE. Identical relative mobilities and homogeneity were found for these enzymes.
Enzyme assay and thermostability
Enzyme activity was measured by incubation of 0.10.5 U glucose isomerase in 200 µl 25 mM TEA (triethanolamine) containing 10 mM MgCl2 at 35°C for 15 min, using 0.1 M xylose or 0.6 M glucose as substrate to produce xylulose or fructose. The reaction was terminated with 50% trichloroacetic acid (TCA), then 70% H2SO4(0°C) containing 100 µl 2.4% Cys-HCl and 100 µl 0.12% alcohol-carbazole was added immediately. Then the mixture was incubated at 25°C for 30 min. The production of xylulose or fructose cooled in ice water was monitored in cuvettes with a 1 cm light path at 560 nm with a spectrophotometer. Heat inactivation profiles of wild-type and mutant glucose isomerase were determined at 80°C. A 0.5 mg sample/ml solution in 50 mM TrisHCl containing 100 mM MgCl2 (pH 7.0) was heated, and every 30 min an aliquot was removed and immediately placed on ice, and the residual activity was assayed at 35°C. One unit was defined as the amount of enzyme that produces 1 µmol xylulose or fructose per minute under these conditions.
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Results and discussion |
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After comparing the primary sequences of GI from thermophilic bacteria with those of other GIs and S.M33GI, we found Pro138 was specific in thermophilic bacteria. So we replaced the corresponding amino acid, which was Gly in S.M33GI, with Pro and carried out space simulation. Molecular modelling of the mutants of S.M33 XI is based on the structural model of wild-type XI at 1.9 Å resolution (Zhu et al., 1996). Figure 1A
illustrates the overall organization of wild-type S.M33 XI around Gly138. It is very clear that there is more space for the proline side chain from this structure. When Gly138, at the turn from residue 130 to 143, which is close to the Trp137 residue of the catalytic core, is replaced by Pro, the pyrrolidine ring of Pro138 can fill perfectly the empty hole left by Gly which has no side chain, as shown in Figure 1B
. Indeed, G138P is more stable than wild-type XI: its half-life increases by 1 time, as shown in Figure 2
, and its specific activity is 12.71 U/mg, which is about 95% of the wild-type enzyme (13.27 U/mg). The highest thermostability is found in the double mutant, G138P/G247D, whose half-life is even higher than that of the G138P mutant enzyme, when using xylose as a substrate. In addition, the thermostability of G138P and G138P/G247D were also higher than the wild type, when glucose is used as a substrate. Gly247 is the last residue in a ß-sheet spanning residues 242 to 247, and lies in the vicinity of the active core. The result is that the specific activity of G247D is 17.65 U/mg, an increase of 33% but its thermostability decreases a great deal. Possibly the introduction of Asp247 changes the charge transfer system surrounding the active site, and as a result influences its activity.
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We suggest that the GlyPro substitution may also improve the thermostability of other glucose isomerases. This conclusion is supported by the following observations. First, comparison of amino acid sequences of XI from 19 bacterial sources reveals that D
E, Q
H and G
P substitutions are much more prominent in XI from Thermus thermophilus (Bhosale et al., 1996
), which is the most thermostable of known glucose isomerases (Dekker et al., 1991
). Interestingly, Gly138 is conserved in these glucose isomerases except for XI from T.thermophilus, whose amino acid at 137 (corresponding to Gly138 in S.M33 XI) is just Pro. Second, comparisons of available XI crystal structures from the PDB show that XI's from Streptomyces rubiginosus (Carrell et al., 1989
) and Arthrobacter N.R.R.L.B3728 (Smith et al., 1991
) display similar effects generated by the Gly138Pro replacement in S.M33 XI. Thus, comparisons of structure and function among homological proteins are helpful to molecular design in protein engineering. In addition, as for oligomeric enzymes, the focus of enhancing heat stability is usually enhancing interaction between subunits. Our findings also indicate that the Gly138Pro substitution in a turn can increase the thermostability of oligomeric enzymes.
The growth behavior of the crystal of the mutant expressed in E.coli seems to differ greatly from that of the wild-type crystal. At present we have not obtained a crystal with high enough X-ray resolution, so it is hard to tell just what has been changed in the structure due to these mutations. We can only postulate the changes using simulation analysis of the wild-type GI. The crystal structure may reveal why we have such results. The crystal study of the mutant enzymes GI (G138P,G247D and G138P/G247D) is in progress.
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Acknowledgments |
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Notes |
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4 These authors contributed equally to this work.
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References |
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Received March 9, 1999; revised May 7, 1999; accepted May 7, 1999.