title: 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.
authors.affiliation: Howard Hughes Medical Institute and Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana-Champaign , 600 S. Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.affiliation: Howard Hughes Medical Institute and Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana-Champaign , 600 S. Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Wilfred A
authors.initials: WA
authors.lastname: van der Donk
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D010455:Peptides; C015102:dehydroalanine; D010088:Oxidoreductases; D000409:Alanine