Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose
Development of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst nece...
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| Format: | Article |
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MDPI AG
2022-10-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/20/12540 |
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| author | Evgeniy A. Zayats Ilya V. Fateev Maria A. Kostromina Yulia A. Abramchik Dmitry D. Lykoshin Daria O. Yurovskaya Vladimir I. Timofeev Maria Ya. Berzina Barbara Z. Eletskaya Irina D. Konstantinova Roman S. Esipov |
| author_facet | Evgeniy A. Zayats Ilya V. Fateev Maria A. Kostromina Yulia A. Abramchik Dmitry D. Lykoshin Daria O. Yurovskaya Vladimir I. Timofeev Maria Ya. Berzina Barbara Z. Eletskaya Irina D. Konstantinova Roman S. Esipov |
| author_sort | Evgeniy A. Zayats |
| collection | DOAJ |
| description | Development of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst necessary for this approach, ribokinase from <i>Escherichia coli</i> (<i>Eco</i>RK), has a very low activity towards D-arabinose, making the synthesis using the state-of-art native enzyme technologically unfeasible. Here, we report the results of our enzyme design project, dedicated to engineering a mutant form of <i>Eco</i>RK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low <i>Eco</i>RK activity towards arabinose and select feasible mutations. Enzyme assay and kinetic studies have shown that the A98G mutation has caused a large 15-fold increase in k<sub>cat</sub> and 1.5-fold decrease in K<sub>M</sub> for arabinose phosphorylation. As a proof of concept, we have performed the cascade synthesis of 2-chloroadenine arabinoside utilizing the A98G mutant with 10-fold lower amount of enzyme compared to the wild type without any loss of synthesis efficiency. Our results are valuable both for the development of new technologies of synthesis of modified nucleosides and providing insight into the structural reasons behind <i>Eco</i>RK substrate specificity. |
| first_indexed | 2024-03-09T20:06:11Z |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T20:06:11Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-bdf401294f1d4b3f8d79cf83b01d4c6a2023-11-24T00:33:11ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-10-0123201254010.3390/ijms232012540Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinoseEvgeniy A. Zayats0Ilya V. Fateev1Maria A. Kostromina2Yulia A. Abramchik3Dmitry D. Lykoshin4Daria O. Yurovskaya5Vladimir I. Timofeev6Maria Ya. Berzina7Barbara Z. Eletskaya8Irina D. Konstantinova9Roman S. Esipov10Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaShemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya St. 16/10, 117997 Moscow, RussiaDevelopment of efficient approaches for the production of medically important nucleosides is a highly relevant challenge for biotechnology. In particular, cascade synthesis of arabinosides would allow relatively easy production of various cytostatic and antiviral drugs. However, the biocatalyst necessary for this approach, ribokinase from <i>Escherichia coli</i> (<i>Eco</i>RK), has a very low activity towards D-arabinose, making the synthesis using the state-of-art native enzyme technologically unfeasible. Here, we report the results of our enzyme design project, dedicated to engineering a mutant form of <i>Eco</i>RK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low <i>Eco</i>RK activity towards arabinose and select feasible mutations. Enzyme assay and kinetic studies have shown that the A98G mutation has caused a large 15-fold increase in k<sub>cat</sub> and 1.5-fold decrease in K<sub>M</sub> for arabinose phosphorylation. As a proof of concept, we have performed the cascade synthesis of 2-chloroadenine arabinoside utilizing the A98G mutant with 10-fold lower amount of enzyme compared to the wild type without any loss of synthesis efficiency. Our results are valuable both for the development of new technologies of synthesis of modified nucleosides and providing insight into the structural reasons behind <i>Eco</i>RK substrate specificity.https://www.mdpi.com/1422-0067/23/20/12540enzyme genetic improvementrational designactive site modificationsite-directed mutagenesisribokinasecascade synthesis |
| spellingShingle | Evgeniy A. Zayats Ilya V. Fateev Maria A. Kostromina Yulia A. Abramchik Dmitry D. Lykoshin Daria O. Yurovskaya Vladimir I. Timofeev Maria Ya. Berzina Barbara Z. Eletskaya Irina D. Konstantinova Roman S. Esipov Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose International Journal of Molecular Sciences enzyme genetic improvement rational design active site modification site-directed mutagenesis ribokinase cascade synthesis |
| title | Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose |
| title_full | Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose |
| title_fullStr | Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose |
| title_full_unstemmed | Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose |
| title_short | Rational Mutagenesis in the Lid Domain of Ribokinase from <i>E. coli</i> Results in an Order of Magnitude Increase in Activity towards D-arabinose |
| title_sort | rational mutagenesis in the lid domain of ribokinase from i e coli i results in an order of magnitude increase in activity towards d arabinose |
| topic | enzyme genetic improvement rational design active site modification site-directed mutagenesis ribokinase cascade synthesis |
| url | https://www.mdpi.com/1422-0067/23/20/12540 |
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