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

ABSTRACT: 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. NpnJA 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.

SUBMITTER: Yang X

PROVIDER: S-EPMC4599312 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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

Yang Xiao X van der Donk Wilfred A WA

Journal of the American Chemical Society 20150924 39

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. NpnJA reduces dehydroalanine to D-Ala using NAPDH as cosubstrate ...[more]

PMID: 26361061

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