1 National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 212 Kannondai, Tsukuba, Ibaraki 305-8642, 3 Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, 4 National Institute for Advanced Interdisciplinary Research, Ministry of International Trade and Industrial Science, Tsukuba, Ibaraki 305-8562, 5 National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries, 212 Kannondai, Tsukuba, Ibaraki 305-8602, 6 Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 and 7 Institute of Applied Biochemistry, University of Tsukuba, 111 Tennoodai, Tsukuba, Ibaraki 305-8572, Japan
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Abstract |
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Keywords: Cellulomonas fimi/Cex,chimeric xylanase/family 10 xylanase/module/Streptomyces olivaceoviridis
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Introduction |
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The enzyme FXYN from Streptomyces olivaceoviridis E-86 was selected as one of the parent enzymes since we have been successful in crystallizing the intact FXYN (Fujimoto et al., 1997, 2000
) and also in isolating its gene (Kuno et al., 1998
). In addition, the substrate specificity of the FXYN has been well characterized (Kusakabe et al., 1977
, 1983
; Yoshida et al., 1990
, 1994
; Matsuo et al., 1991
). In a previous paper (Kaneko et al., 1999
), we reported the effect of the replacement of module M10 because substrate binding amino acid residues had been identified by co-crystallization of Cex with an inhibitor (White et al., 1996
) as being located in modules M4, M6, M7, M10, M15, M19 and M20 (Sato et al., 1999
). In the present investigation, we selected modules M4 and M5 for shuffling. Modules M4 and M5 are one of the additional helices of the basic TIM barrel and are arranged in a loop to form part of the substrate-binding cleft of the enzyme (Figure 1
). It is an important module for family F/10 xylanases because it contains amino acids which form the 1 to 3 sites of the substrate-binding cleft (Figure 2
). To endeavour to understand the function of these modules, we constructed chimeric xylanases. In this paper, the function of modules M4 and M5 and the correlation of these results with those from module M10 are described.
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Materials and methods |
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The module arrangement of chimeric xylanases is shown in Figure 3. The catalytic domains of the FXYN and Cex xylanases were separately subcloned into the pQE60 vector (Qiagen, Hilden, Germany). Construction of the chimeras was performed by the polymerase chain reaction (PCR) using overlapping primers at their respective module boundaries (Kaneko et al., 1999
). Briefly, DNA fragments from the FXYN coding modules M1 to M3 or M1 to M4 and modules M5 to M22 or M6 to M22 were amplified by PCR (Takara LA Taq, Takara Shuzo, Shiga, Japan). Each of the 25 amplification cycles consisted of denaturation at 98°C for 1 min and annealing and primer extension at 72°C for 1 min. DNA coding for module M4, module M5 or modules M4 and M5 of the Cex gene, with an overlapping region for the FXYN gene, were synthesized. The 10 bp overlapping regions of the primers were designed to be complementary at their respective module boundaries. The first round of PCR products were separated by agarose gel electrophoresis, followed by gel extraction, and used for the second round PCR without primers. Each of the 20 amplification cycles consisted of denaturation at 98°C for 1 min, annealing at 60°C for 25 min and primer extension at 72°C for 5 min. The strands having matching sequences at their respective module boundaries overlapped and acted as primers for each other. On the third round of PCR, the combined fragment was amplified by PCR primers with 25 cycles of shuttle PCR with denaturation at 98°C for 1 min and annealing and primer extension at 72°C for 1 min. The PCR products were subcloned into the plasmid pCR2.1 using the Original TA Cloning Kit and INV
F' cells (Invirogen, Carlsbad, CA), then the insert was sequenced with an automated DNA sequencer (Model 310, Applied Biosystems, Foster City, CA).
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For expression in E.coli and purification of the FXYN, Cex, FCF-C4, FCF-C5, FCF-C4,5 and FCFCF-C4,10, the pET expression system (Novagen, Madison, WI) was employed. Each gene was individually inserted into the pET28a vector and the enzyme was expressed as a fusion protein consisting of the enzyme with a carboxyl-terminal tag with six histidine residues attached. Plasmids were used to transform E.coli BL21 (DE3) and the transformants were cultivated at 25°C in LB medium (1 l) containing kanamycin (20 µg/ml) until the optical density reached 0.4 at 600 nm. After the addition of isopropyl-1-thio-ß-D-galactoside (IPTG) to give a final concentration of 1 mM, the culture was incubated at 25°C for 24 h. The expressed enzymes were purified with a HisTrap chelating column (Pharmacia, Uppsala, Sweden). The enzyme was eluted from the column as a homogeneous protein by SDSPAGE and the relevant fractions were pooled and dialysed against deionized water. The recovery of the FXYN, Cex, FCF-C4, FCF-C5 and FCFCF-C4,10 from 1 l of culture was 4.1, 0.1, 13.0, 0.04 4.4 mg, respectively.
Circular dichroism and steady-state kinetic studies
The circular dichroism spectra of the FXYN, Cex, FCF-C4, FCF-C5 and FCFCF-C4,10 enzymes were acquired using the conditions reported previously (Kaneko et al., 1999; Kuno et al., 1999
). Steady-state kinetics were investigated as reported previously (Kaneko et al., 1999
; Kuno et al., 1999
). Briefly, the reaction mixture containing the substrate at various concentrations in 25% McIlvaine buffer (a mixture of 0.1 M citric acid and 0.2 M Na2HPO4, pH 7.0), containing 0.05% bovine serum albumin (BSA), was incubated at 30°C for 5 min before 50 µl of enzyme solution were added. The amount of p-nitrophenol released was determined by monitoring the absorbance at 400 nm with a spectrophotometer (DU-7400; Beckman, Palo Alto, CA). p-Nitrophenyl-ß-D-xylobioside (PNP-X2) was synthesized by the method described in a previous paper (Takeo et al., 1995
). The xylobiose used in the synthesis was purified from `Xylobiose Mixture' (Suntory, Osaka, Japan). p-Nitrophenyl-ß-D-cellobioside (PNP-G2) was kindly donated by Yaizu Suisan (Yaizu, Japan).
Enzymatic properties
The effects of pH on the activity and stability of the various xylanases were investigated using a series of McIlvaine buffers (0.2 M Na2HPO40.1 M citric acid) from pH 4.0 to 8.0 and AtkinsPantin buffers (0.2 M boric acid + 0.2 M KCl0.2 M Na2CO3) from pH 8.0 to 10.5. The activities of the xylanases were assayed as follows. Each assay mixture contained 0.5 ml of a 2 mM PNP-X2 solution, 0.4 ml of buffer and 0.1 ml of enzyme solution. The reactions were performed at 45°C for 10 min whereupon they were stopped by the addition of 1.0 ml of 0.2 M Na2CO3 solution and the amount of p-nitrophenol (PNP) released was then determined at 408 nm. For determinations of the pH stabilities of the xylanases, the enzymes were pre-incubated in the absence of substrate at 30°C for 60 min and the residual activity was assayed at 45°C and pH 5.7. The effect of temperature on the activity of the xylanases was examined using a series of water-baths ranging from 30 to 70°C. Xylanase activity was measured at various temperatures at pH 5.7. For temperature-stability measurements of the various xylanases, the enzymes were pre-incubated at various temperatures at pH 7.0 for 60 min and the residual activity was then determined at 45°C and pH 5.7.
Xylan hydrolysis by chimeric xylanases
A reaction mixture containing 150 µl of McIlvaine buffer (pH 7.0), 50 µl of 1% (v/w) BSA and 250 µl of 1% soluble birchwood xylan solution was equilibrated at 30°C for 5 min and reactions were initiated by the addition of 50 µl of the enzyme (the final concentrations of FXYN, Cex, FCF-C4, FCF-C5 and FCF-C4, 10 were 0.012, 0.035, 0.023, 0.025 and 0.042 mg/ml, respectively. The amount of enzyme was adjusted by the production of reducing sugar from soluble birchwood xylan. After 0, 0.25, 0.5, 0.75, 1, 1.5, 3, 6, 12 and 24 h incubations, the reaction was terminated by boiling for 5 min. The reducing power generated from the soluble xylan was determined by the SomogyiNelson method (Somogyi, 1952). The reaction products were also analyzed using an HPAECPAD system (Dionex International Subsidiaries, Osaka, Japan).
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Results |
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The overall similarity of the catalytic domains between the FXYN and Cex enzymes is 49%. As shown in Figure 1, the positions of modules M4 and M5 in the catalytic domain are in close proximity to catalytic amino acids such as E128 and E236 of the FXYN or to E127 and E233 of the Cex. Some of the amino acid residues forming the binding subsite are included in modules M4 and M5. The importance of these amino acids in substrate binding has been investigated by site-directed mutagenesis (Charnock et al., 1997
, 1998
). Therefore, we selected modules M4 and M5 for a shuffling investigation and chimeras FCF-C4, FCF-C5 and FCF-C4,5 were constructed (Figure 3
). The activities of the constructed chimeric enzymes were detected on Remazole Brilliant Bluexylan containing LB agar plates (data not shown). However, FCF-C4,5 was purified as inactive protein. The loss of activity of the enzyme during the purification step resulted from the high instability of the enzyme. Therefore, only FCF-C4 and FCF-C5 were purified and subjected to further investigations. Circular dichroism spectra obtained from the enzymes indicated that the constructed chimeric enzymes folded in the same conformational manner as the parent enzymes (Figure 4
). The enzymatic properties of the FCF-C4 and FCF-C5 chimeras are shown in Figure 5
. The optimum pHs of both chimeric enzymes were shifted to a slightly more acidic pH (from pH 6 of parental enzymes to pH 5), but the pH stability of the enzymes remained unchanged (Figure 5A and C
). The optimum temperature of both FCF-C4 and FCF-C5 was 55°C, which was slightly lower than the 60°C observed for the parental FXYN and Cex enzymes (Figure 5B
) and the thermostabilities of the chimeric enzymes were also slightly decreased compared with the parental enzymes (Figure 5D
). Kinetic data were collected for both FCF-C4 and FCF-C5 along with parental FXYN and Cex using PNP-G2 and PNP-X2 as the substrates (Table I
). Compared with the Cex, the FXYN enzyme displays much lower levels of activity towards both of the substrates. The FXYN enzyme displays kcat/Km values ~70-fold (for PNP-G2) and ~30-fold (for PNP-X2) lower than the corresponding Cex values. Because the majority of the chimeric enzymes FCF-C4 and FCF-C5 originated from the FXYN enzyme, the kinetic parameters of these enzymes would be expected to be closer to those of the parental FXYN rather than those of the Cex. The Km values of FCF-C4 and FCF-C5 for PNP-G2 were 112 and 96 mM, respectively, almost the same as that of the FXYN (97 mM). The kcat value of FCF-C5 for PNP-G2 (1.7 s1) was also similar to the value of FXYN (2.2 s1); however, surprisingly, the kcat value of FCF-C4 for PNP-G2 was significantly higher (7.0 s1). The Km and kcat values of FCF-C4 and FCF-C5 for PNP-X2 were 0.88 mM and 21 s1 and 2.4 mM and 33 s1, respectively. The kcat/Km value of FCF-C4 was unchanged relative to FXYN. However, the kcat/Km value of FCF-C5 was slightly lower than that of FXYN, primarily owing to a reduction in the kcat value. The activities of the chimeric xylanases for the native substrates were also determined (Figure 6
). When twice the amounts of FCF-C4 and FCF-C5 were used relative to FXYN, the enzyme activities were almost the same, indicating that the kcat values of the chimeric xylanases were almost half those of the FXYN enzyme. However, the mode of action of xylan hydrolysis by the chimeric enzymes was not altered, as evidenced by the analysis of the hydrolysis products by HPEACPAD (data not shown).
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We previously reported the characterization of the chimera FCF-C10 in which module M10 of the FXYN was replaced with that of the Cex enzyme (Kaneko et al., 1999). Both the kcat and Km values observed for FCF-C10 were decreased by a factor of 10 relative to FXYN (Kaneko et al., 1999
). To investigate the synergistic effects of the modules, module M10 of FCF-C4 was replaced with that of the Cex. The constructed chimera, FCFCF-C4,10, was expressed in an active form. FCFCF-C4,10 was purified and subjected to the same investigations as FCF-C4 and FCF-C5. Circular dichroism spectra of FCFCF-C4,10 indicated that the enzyme folded in the same manner as the parent enzymes (Figure 4
). The enzymatic properties of the FCFCF-C4,10 chimera are depicted in Figure 5
. The optimum pH of the chimeric enzyme was almost the same as that of the parental enzymes, but a difference in the pH stabilities of FCFCF-C4,10 and the FXYN was observed (Figure 5A and C
). The stability of FCFCF-C4,10 in the mid-range of pH values was slightly lower in comparison with the other enzymes. Both the optimum temperature and the thermostability range of the FCFCF-C4,10 chimera were slightly reduced relative to the parental enzymes but were similar to those of the FCF-C4 and FCF-C5 enzymes (Figure 5B and D
). The kinetic data obtained for the FCFCF-C4,10 chimera toward PNP-G2 and PNP-X2 are shown in Table I
. Compared with the FCF-C4 chimera, FCFCF-C4,10 displays a lower activity towards both substrates. FCFCF-C4,10 displays a kcat/Km value ~2.5-fold lower for PNP-G2 and ~2.8-fold lower for PNP-X2 relative to the FCF-C4 chimera. The kinetic parameters of the FCFCF-C4,10 enzyme were closer to those of FCF-C10 than to those of FCF-C4. The Km value for FCFCF-C4,10 toward PNP-G2 was 46 mM, which was closer to that of FCF-C10 (64 mM) than to that for FCF-C4 (112 mM). The kcat value of FCFCF-C4,10 toward PNP-G2 (1.2 s1) was also similar to the value observed for FCF-C10 (1.8 s1). The Km value of FCFCF-C4,10 toward PNP-X2 was 0.14 mM, close to that observed for FCF-C10 (0.24 mM) and 6.3-fold lower than that seen for FCF-C4 (0.88 mM). The kcat value obtained for FCFCF-C4,10 toward the substrate PNP-X2 (1.2 s1) was also reduced to a level 17.5-fold lower than that seen with FCF-C4 (21 s1). The activities of the FCFCF-C4,10 chimera toward the native substrates were also determined (Figure 6
). The enzyme activities of FCFCF-C4,10 were almost the same as those of the other enzymes when using twice the amount of enzyme as FCF-C4, indicating that the kcat value of FCFCF-C4,10 was almost half that of FCF-C4. However, the mode of action of xylan hydrolysis by the chimeric enzymes was unchanged, as evidenced by the analysis of the hydrolysis products by HPEACPAD (data not shown).
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Discussion |
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When the module M10 of FCF-C4 was replaced with that of Cex, the Km value for PNP-X2 was also decreased from 0.88 to 0.14 mM together with kcat value from 21 to 1.2 s1. There was a similar situation when module M10 of FXYN was replaced with that of Cex. Therefore, the effect of module M10 was almost the same for FXYN and FCF-C4 so that in FCFCF-C4,10, the effects of replacement of module M4 and M10 were almost additive.
In conclusion, our results indicate that in xylanases, module M4, including the substrate-binding residues (E44, N45 and K48 in FXYN), is not only related directly to enzyme activity but also interacts with adjacent modules which are involved in forming the substrate-binding cleft. A similar finding was apparent in the case of module M10 replacement (Kaneko et al., 1999). The double replacement of modules M4 and M10 displayed additive effects in Km and kcat for replacing modules M4 and M10 individually. These results indicate that module shuffling or exon suffling is a possible mechanism in the evolution of proteins.
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Notes |
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Acknowledgments |
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References |
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Received June 30, 2000; revised October 26, 2000; accepted October 30, 2000.