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Bioinformatic analysis and molecular modeling reveal mutation bD484N to stabilize penicillin acylase and improve its catalytic performance in alkaline mediumстатья
Информация о цитировании статьи получена из
Web of Science
Статья опубликована в журнале из списка Web of Science и/или Scopus
Дата последнего поиска статьи во внешних источниках: 27 мая 2015 г.
- Авторы: Panin N., Suplatov D., Kirilin E., Shcherbakova T., Kudravtsev P., Švedas V.
- Журнал: FEBS Journal
- Том: 280
- Номер: 1
- Год издания: 2013
- Издательство: Blackwell Publishing Inc.
- Местоположение издательства: United Kingdom
- Первая страница: 614
- DOI: 10.1111/febs.12340
- Аннотация: Rational design strategy based on bioinformatic analysis of Ntn-hydrolase family enzymes and molecular modeling of potential mutants was applied to improve stability and catalytic performance of penicillin acylase from Escherichia coli. Recently developed method of bioinformatic analysis [1] and corresponding computer program Zebra [http://biokinet.belozersky.msu.ru/zebra] were applied to identify subfamily-specific positions that were supposed to be responsible for discrimination of functional properties of Ntn-hydrolases and considered as hotspots for rational design of penicillin acylase mutants. Molecular dynamics was used to simulate pH-dependent inactivation of wild type enzyme and its mutants. Subfamily-specific position bD484 was identified as a key element of the buried side chain interaction network, which collapse at alkaline pH disturbs a native protein conformation. This crucial residue was chosen as a hotspot for mutation to engineer enzyme variant stable in alkaline medium: single stabilizing substitution bD484N has been proposed by bioinformatic analysis of homologous Ntn-hydrolases with different pH stability. The bD484N mutant was expressed, purified and characterized experimentally. The bD484N mutation substantially stabilized penicillin acylase in alkaline medium (10-fold improvement at pH 10.0) however even more important was enzyme stabilization to inactivation at high substrate concentration. Observed stabilization effects allowed to improve catalytic performance of penicillin acylase at enzymatic peptide synthesis in aqueous medium (where due to the high pKa values of the amino group of free amino acids alkaline pH should be used in order to have external nucleophiles in their reactive form) and leaded to 5-fold increased yield of preparative D-phenylglycine-derived peptide synthesis from equimolar substrate mixtures compared to the wild type enzyme.
- Добавил в систему: Суплатов Дмитрий Андреевич