Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR
Abstract XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type...
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Nature Portfolio
2024-04-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-024-57819-z |
| _version_ | 1797219856772235264 |
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| author | Tomoka Nakamura Teisuke Takita Kohei Kuwata Kimihiko Mizutani Bunzo Mikami Satoshi Nakamura Kiyoshi Yasukawa |
| author_facet | Tomoka Nakamura Teisuke Takita Kohei Kuwata Kimihiko Mizutani Bunzo Mikami Satoshi Nakamura Kiyoshi Yasukawa |
| author_sort | Tomoka Nakamura |
| collection | DOAJ |
| description | Abstract XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type XynR (WT) > 15 other variants. In this study, we sought to elucidate the mechanisms underlying the variable activity of these different variants. Crystallographic analysis revealed that the Ca2+ ion near position 315 in WT was absent in the T315Q variant. We accordingly hypothesized that the enhancement of alkaliphily in T315Q, and probably also in the T315H, T315N, and T315S variants, could be ascribed to an activity-stability trade-off associated with a reduction in stability due to the lack of this Ca2+ ion. Consistent with expectations, the alkaline resistance of T315H, T315N, T315Q, and T315S, evaluated through the pH-dependence of stability at 0 mM CaCl2 under alkaline conditions, was found to be lower than that of WT: the residual activity at pH 11 of WT was 78% while those of T315H, T315N, T315Q, and T315S were 0, 9, 0, and 43%, respectively. In addition, the thermostabilities of these four variants, as assessed using the denaturing temperatures (T m) at 0 mM CaCl2 based on ellipticity at 222 nm in circular dichroism measurements, were lower than that of WT by 2–8 °C. Furthermore, the T m values of WT and variants at 5 mM CaCl2 were higher than those at 0 mM CaCl2 by 6–11 °C. Collectively, our findings in this study indicate that mutation of the T residue at position 315 of XynR to H, N, Q, and S causes an increase in the alkaliphily of this enzyme, thereby reducing its stability. |
| first_indexed | 2024-04-24T12:40:18Z |
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| id | doaj.art-3d4e5e5160a24dd5b0c1d6cd6d9a1d4f |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-24T12:40:18Z |
| publishDate | 2024-04-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-3d4e5e5160a24dd5b0c1d6cd6d9a1d4f2024-04-07T11:14:24ZengNature PortfolioScientific Reports2045-23222024-04-0114111210.1038/s41598-024-57819-zActivity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynRTomoka Nakamura0Teisuke Takita1Kohei Kuwata2Kimihiko Mizutani3Bunzo Mikami4Satoshi Nakamura5Kiyoshi Yasukawa6Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto UniversityDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto UniversityDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto UniversityDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityDepartment of Life Science and Technology, Tokyo Institute of TechnologyDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto UniversityAbstract XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type XynR (WT) > 15 other variants. In this study, we sought to elucidate the mechanisms underlying the variable activity of these different variants. Crystallographic analysis revealed that the Ca2+ ion near position 315 in WT was absent in the T315Q variant. We accordingly hypothesized that the enhancement of alkaliphily in T315Q, and probably also in the T315H, T315N, and T315S variants, could be ascribed to an activity-stability trade-off associated with a reduction in stability due to the lack of this Ca2+ ion. Consistent with expectations, the alkaline resistance of T315H, T315N, T315Q, and T315S, evaluated through the pH-dependence of stability at 0 mM CaCl2 under alkaline conditions, was found to be lower than that of WT: the residual activity at pH 11 of WT was 78% while those of T315H, T315N, T315Q, and T315S were 0, 9, 0, and 43%, respectively. In addition, the thermostabilities of these four variants, as assessed using the denaturing temperatures (T m) at 0 mM CaCl2 based on ellipticity at 222 nm in circular dichroism measurements, were lower than that of WT by 2–8 °C. Furthermore, the T m values of WT and variants at 5 mM CaCl2 were higher than those at 0 mM CaCl2 by 6–11 °C. Collectively, our findings in this study indicate that mutation of the T residue at position 315 of XynR to H, N, Q, and S causes an increase in the alkaliphily of this enzyme, thereby reducing its stability.https://doi.org/10.1038/s41598-024-57819-z |
| spellingShingle | Tomoka Nakamura Teisuke Takita Kohei Kuwata Kimihiko Mizutani Bunzo Mikami Satoshi Nakamura Kiyoshi Yasukawa Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR Scientific Reports |
| title | Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR |
| title_full | Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR |
| title_fullStr | Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR |
| title_full_unstemmed | Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR |
| title_short | Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR |
| title_sort | activity stability trade off observed in variants at position 315 of the gh10 xylanase xynr |
| url | https://doi.org/10.1038/s41598-024-57819-z |
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