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Bioinformatic analysis of penicillin-binding proteins identified amino acid residues responsible for substrate specificity of D-aminopeptidase from Ochrobactrum anthropiстатья
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: Khaliullin I., Suplatov D., Tokunan K., Himi M., Asano Y., Švedas V.
- Журнал: FEBS Journal
- Том: 280
- Номер: 1
- Год издания: 2013
- Издательство: Blackwell Publishing Inc.
- Местоположение издательства: United Kingdom
- Первая страница: 592
- DOI: 10.1111/febs.12340
- Аннотация: D-aminopeptidase from Ochrobactrum anthropi (DAP) is a unique member of penicillin-binding proteins (PBP) family that converts mainly D-alanine containing peptides, amides, and esters with high D-stereospecificity. Consequently, in this work we aimed to study structure-function relationship of substrate recognition in DAP and rationally design its specificity toward substrates with bulky side-chain residues – D-phenylalanine and D-leucine derivatives. Recently developed bioinformatic analysis [1,2] of penicillin-binding proteins (PBP) family was used to identify subfamily-specific positions (SSPs) that are conserved within functional subfamilies of PBP, but different between subfamilies and therefore expected to be responsible for functional diversity among homologs. Specific positions that participate in formation of acylgroup binding subsite were selected as hotspots to rationally design specificity of DAP toward selected substrates by introducing amino acid types observed in other subfamilies. Molecular modeling was used to screen the corresponding in silico library of DAP mutants and select the best variants by evaluating their ability to stabilize near-to-attack conformation of the substrate in the active site cleft. Selected triple mutant was purified and characterized experimentally to successfully hydrolyze aromatic and branched aliphatic D-amino acid derivatives in a full agreement with computational predictions. The results outline perspectives for bioinformatic analysis of subfamily-specific positions as a systematic tool to understand structure-function relationship in enzymes. Selection of subfamily-specific positions as hotspots for rational design of enzyme properties is suggested as a new protein engineering strategy.
- Добавил в систему: Суплатов Дмитрий Андреевич