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

Susan L. Clugston, Stephan A. Sieber, Mohamed A. Marahiel, Christopher T. Walsh

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80 Scopus citations

Abstract

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 (DCL). To validate this prediction and establish that the C5 domain of tyrocidine synthetase is indeed DCL, 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 DCL 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.

Original languageEnglish
Pages (from-to)12095-12104
Number of pages10
JournalBiochemistry
Volume42
Issue number41
DOIs
StatePublished - 21 Oct 2003
Externally publishedYes

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