1 Tochigi Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497 and 2 Department of Biotechnology and Biomaterial Chemistry, Graduate School of Engineering, Nagoya University, Chikusa-ku, Nagoya 464-8603, Japan
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Abstract |
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Keywords: Bacillus/cellulase/intrahelix/ion pair/salt bridge/thermostability
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Introduction |
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Recently, we found a thermostable, alkaline Egl (Egl-237) from the alkaliphilic Bacillus isolate KSM-S237 (Hakamada et al., 1997) and sequenced the cloned gene of the enzyme (Hakamada et al., 2000
). The deduced amino acid sequence of Egl-237 showed high homology with those of Egl-K (Ozaki et al., 1990
) and Egl-64 (Sumitomo et al., 1992
). Based on the difference of a few amino acids, we constructed several chimeric genes from Egl-64 and Egl-237, and Lys179 and Lys194 residues in Egl-237, not conserved in Egl-64, were found to contribute to thermostability after site-directed mutagenesis. As a result, the double mutation Asn179
Lys (NK)/Asp194
Lys (DK) (NK/DK) significantly improved thermostability, the level of which was as high as that of the wild-type Egl-237 (Hakamada et al., 2001
). One of the most striking features of thermostable enzymes, when compared with mesophilic enzymes, is the decrease in the number of Lys residues, which are mainly replaced with Arg and Glu residues (Argos et al., 1979
; Davies et al., 1993
; Shirai et al., 1997a
). However, the thermal stabilization of mesophilic Egl-64 by the additional Lys residues instead of Arg residues is clearly contrary to these results with thermophilic enzymes. In spite of the gained basic Lys residue(s), the pI value of the mutant Egl-64 enzymes estimated by electrofocusing was slightly higher than or similar to that of the wild-type level. This suggests that the thermal stabilization achieved with the mutations involves the formation of new ion pairs, such as salt bridges, with some carboxylate residues localized on the enzyme surfaces. Although the 3D structures of glycosyl hydrolase family 5 cellulases have been determined (Ducros et al., 1995
; Sakon et al., 1996
; Davies et al., 1998
), their amino acid sequences and catalytic properties were too different from those of Egl-64 and Egl-237 in the same family to analyze the roles of the thermostabilizing Lys residues. Recently, we succeeded in crystallizing (Shirai et al., 1997b
) and determining the 3D structure of the truncated Egl-K, designated Egl-Kt (Ozaki et al., 1995
), similar to the two alkaline Bacillus Egls. Here we show the precise involvement of the specific Lys residues in and the mechanisms for the thermal stabilization of Egl-64 by homology modeling and implicating by molecular dynamics (MD) simulation.
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Materials and methods |
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The alkaliphilic Bacillus sp. strain KSM-64 of our stock cultures was used as the source of the gene encoding Egl-64 (Sumitomo et al., 1992). Constructed plasmids were used individually for transformation of B.subtilis ISW1214 to exoproduce wild-type, NK, DK or NK/DK mutant enzyme in the medium described previously (Sumitomo et al., 1995
; Hakamada et al., 2001
).
Purification and assay of enzymes
The recombinant enzymes produced by B.subtilis were purified by chromatography on a DEAE-Toyopearl 650C column to homogeneity as judged by SDSPAGE. Egl activity was measured at 40°C in 0.1 M glycineNaOH buffer (pH 9.0) with 1.0% CMC as substrate by the dinitrosalicylic acid procedure as described (Hakamada et al., 2000).
Homology modeling of tertiary structures of Egl
The structural models of Egl-64 and its engineered protein (NK/DK) were constructed by the homology modeling method, based on the 3D structure of Egl-Kt (PDB code No. 1go1) and the deduced amino acid sequence of Egl-64 (Sumitomo et al., 1992). All data sets were processed on a Silicon Graphics Indigo2 workstation using the InsightII/Discover software package (Molecular Simulation). MD simulations were calculated in an AMBER forcefield, where a dielectric constant
of 1 and the partial atomic charges computed at pH 7 were used. The MD simulations were performed in 4000 fs after linear heating from 0 to 330 K within 1000 fs and the MD trajectory was saved at 20 fs intervals.
Gene accession numbers
The original nucleotide sequence data of Egl-64 have been deposited in the DDBJ, EMBL and GenBank databases under the accession No. M84963.
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Results and discussion |
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The thermostability of the NK, DK and NK/DK mutant enzymes was examined by heating each at various temperatures for 30 min in 0.1 M glycineNaOH buffer (pH 9.0). As shown in Figure 1, the replacement of Asp194 with lysine (DK) thermostabilized Egl-64 significantly at high temperatures, as described (Hakamada et al., 2001
). The stabilizing effect of the double mutation NK/DK was reproducibly cumulative, although the single NK mutation improved the thermostability only marginally. At 70°C, the half-lives (t1/2) due to thermal inactivation increased in the order wild-type Egl-64 (9 min), NK mutant (15 min), DK mutant (38 min) and NK/DK mutant (41 min).
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To allow accurate homology modeling and to understand better the mutagenesis results, the amino acid sequence of Egl-64 was aligned with that of Egl-Kt, as shown in Figure 2. High homology was observed within the suitably aligned sequences of both enzymes (73.6% identity and 94.4% similarity). The secondary structural elements,
-helices and ß-strands, including catalytic residues (Hakamada et al. 2000
), in the 3D structure of Egl-Kt are included in Figure 2
.
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To clarify the mechanism of thermostabilization of Egl-64 by the NK/DK mutation, we built a model of the enzyme, based on the 3D structure of Egl-Kt (PDB code No. 1go1), as shown in Figure 3. Before building the model, the amino acid sequences of both enzymes were appropriately aligned (Figure 2
). The validity of the modeled structure was confirmed using the 3D-1D, XPLORE and PROCHECK programs. Owing to the high sequence homology, the modeled structure of Egl-64 (Figure 3B
) closely resembles the 3D structure of Egl-Kt (Figure 3A
). Essentially, the model of Egl-64 contains an
/ß-barrel core, as in the cases of the 3D structures of neutral Egls belonging to the glycosyl hydrolase family 5 (Ducros et al., 1995
; Sakon et al., 1996
; Davies et al., 1998
).
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In the modeled structure of the NK/DK mutant enzyme, the replacements of Lys residues at positions 179 and 194 form ion pairs with the side chains of Glu175 and Glu190, respectively, yielding double intrahelical ion pairs, as shown in Figure 4. Lys194 appears to interact also with Asp224 occurring on a loop between Asn220 and His226.
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Notes |
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References |
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Received January 29, 2001; revised March 23, 2001; accepted April 10, 2001.