Chirality of Peptide Bond-Forming Condensation Domains in Nonribosomal Peptide Synthetases: The C₅ Domain of Tyrocidine Synthetase is a ^DC_L Catalyst

Nonribosomal peptides (NRP) such as the antibiotic tyrocidine have D-amino acids, introduced by epimerase (E) domains embedded within modules of the enzymatic assembly lines. We predict that the peptide bond-forming condensation (C) domains immediately downstream of E domains are D-specific for the peptidyl donor and L-specific for the aminoacyl acceptor (^DC_L). To validate this prediction and establish that the C₅ domain of tyrocidine synthetase is indeed ^DC_L, the apoT (thiolation) forms of module 4 (TycB₃ AT₄E) and module 5 (TycC ₁ C₅AT₅) were expressed. T₅ was posttranslationally primed with CoASH to introduce the HS-pantetheinyl group and autoaminoacylated with radiolabeled L-Asn* or L-Asp*. Alternate donor substrates were introduced by priming apo AT₄E with synthetically prepared tetrapeptidyl-CoA's differing in the chirality of Phe-4, D-Phe-L-Pro-L-Phe-L-Phe-CoA, and D-Phe-L-Pro-L-Phe-D-Phe-CoA. The tetrapeptidyl-S-T₄ and L-Asp*-S-T₅ were studied for peptide bond formation and chain translocation by C₅ to yield pentapeptidyl*-S-T₅, whose chirality (D-L-L-D-L- vs D-L-L-L-L-) was assayed by thioester cleavage and chiral chromatography of the released pentapeptides*. Only the D-Phe-4 pentapeptidyl-S-T₅ was generated, implying that only D-L-L-D-S-T₄ was utilized, proving C₅ is indeed a ^DC_L catalyst. Furthermore, a mutant with an inactive E domain transferred tetrapeptide only when loaded with D-Phe-4 tetrapeptidyl donor, not L-Phe-4, confirming that in the wild-type assembly line C₅ only transfers D-L-L-L-tetrapeptidyl-S-T ₄ after in situ epimerization by the E domain. These results contrast the observation that C₅ can make both L-Phe-L-Asn and D-Phe-L-Asn when assayed with Phe as the donor substrate. Hence, utilizing an aminoacyl-S-T₄ versus the natural peptidyl-S-T₄ donor produced misleading information regarding the specificity of the condensation domain.