Rational Mutagenesis in the Lid Domain of Ribokinase from E. coli Results in an Order of Magnitude Increase in Activity towards D-arabinose

Autor: 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
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 23, Iss 20, p 12540 (2022)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms232012540
Popis: 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 Escherichia coli (EcoRK), 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 EcoRK with elevated activity towards arabinose. Analysis of the active site structure has allowed us to hypothesize the reasons behind the low EcoRK 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 kcat and 1.5-fold decrease in KM 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 EcoRK substrate specificity.
Databáze: Directory of Open Access Journals
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