Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases.
BACKGROUND
Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate specificity is important for the engineered biosynthesis of new compounds.


RESULTS
We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L- and D-Phe-SNACs as upstream (donor) substrates for the first C domain from tyrocidine synthetase revealed clear D- versus L-selectivity.


CONCLUSIONS
Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate specificity of C domain-catalyzed peptide bond formation.
Ehmann
D E
DE
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Trauger
J W
JW
Stachelhaus
T
T
Walsh
C T
CT
D002384:Catalysis; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003543:Cysteamine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D004758:Enterobactin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D004952:Esters / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism*; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D013237:Stereoisomerism; D013379:Substrate Specificity
D004952:Esters; D009097:Multienzyme Complexes; D021122:Protein Subunits; D004758:Enterobactin; D003543:Cysteamine; D008025:Ligases; C066234:enterobactin synthetase; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; C035180:tyrocidine synthetase