ABSTRACT
Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR-ESI-MS proved more successful for the SCS method. Bio-orthogonal chemistry was used for post-biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε-Alloc-L-lysine) instead of Ala. A second-generation Hoveyda-Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post-biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide-based antibiotics from an expanded amino acid repertoire.
Subject(s)
Amino Acids/chemistry , Biochemistry/methods , Peptides/chemistry , Alanine/chemistry , Amino Acid Substitution , Chromatography, High Pressure Liquid , Escherichia coli/genetics , Lysine/chemistry , Peptides/genetics , Protein Engineering/methods , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Mass, Electrospray IonizationABSTRACT
The proximicins A-C are naturally occurring cytotoxic γ-peptides that contain the unique 4-amino-furan-carboxylic acid. In contrast to the structurally related cytotoxic natural DNA binder netropsin and distamycin, both exhibiting as core building block N-methyl-4-amino-pyrrol-carboxylic acid, no DNA binding was observed for the procimicins. X-ray analysis of crystals of a protected 4-amino-furan-2-carboxylic acid dipeptide revealed a stretched conformation. In contrast, for netropsin and distamycin, sickle-shaped crystal conformations were observed. DFT-calculations elegantly confirm these conformational arrangements. The most stable conformers of the proximicins are linear whereas sickle-shaped conformations are less stable, having higher Gibbs energies. For netropsin, distamycin and the netropsin-proximicin-hybrid a sickle shaped conformation appears energetically favored. The reported results are consistent with the observations that the proximicins A-C do not bind to the DNA and have a different mode of action concerning their cytotoxic activity with respect to netropsin and distamycin.
Subject(s)
Anti-Bacterial Agents/chemistry , Peptides/chemistry , Quantum Theory , Crystallography, X-Ray , Models, Molecular , Netropsin/analogs & derivatives , Netropsin/chemistry , Protein ConformationABSTRACT
New beauvericins have been synthesized using the nonribosomal peptide synthetase BbBEAS from the entomopathogenic fungus Beauveria bassiana. Chemical diversity was generated by in vitro chemoenzymatic and in vivo whole cell biocatalytic syntheses using either a B. bassiana mutant or an E. coli strain expressing the bbBeas gene.
