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Post-translational Introduction of d-Alanine into Ribosomally Synthesized Peptides by the Dehydroalanine Reductase NpnJ

[Image: see text] Ribosomally synthesized peptides are generally limited to l-amino acid building blocks. Given the advantageous properties of peptides containing d-amino acids such as stabilization of certain turns and against proteolytic degradation, methods to introduce d-stereocenters are valuab...

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Detalles Bibliográficos
Autores principales: Yang, Xiao, van der Donk, Wilfred A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2015
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599312/
https://www.ncbi.nlm.nih.gov/pubmed/26361061
http://dx.doi.org/10.1021/jacs.5b05207
Descripción
Sumario:[Image: see text] Ribosomally synthesized peptides are generally limited to l-amino acid building blocks. Given the advantageous properties of peptides containing d-amino acids such as stabilization of certain turns and against proteolytic degradation, methods to introduce d-stereocenters are valuable. Here we report the first in vitro reconstitution and characterization of a dehydrogenase that carries out the asymmetric reduction of dehydroalanine. NpnJ(A) reduces dehydroalanine to d-Ala using NAPDH as cosubstrate. The enzyme displays high substrate tolerance allowing introduction of d-Ala into a range of non-native substrates. In addition to the in vitro reactions, we describe five examples of using Escherichia coli as biosynthetic host for d-alanine introduction into ribosomal peptides. A deuterium-label-based coupled-enzyme assay was used to rapidly determine the stereochemistry of the newly installed alanine.