title: Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst.
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 ((D)C(L)). To validate this prediction and establish that the C(5) domain of tyrocidine synthetase is indeed (D)C(L), the apoT (thiolation) forms of module 4 (TycB(3) AT(4)E) and module 5 (TycC(1) C(5)AT(5)) were expressed. T(5) 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 AT(4)E 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-T(4) and L-Asp-S-T(5) were studied for peptide bond formation and chain translocation by C(5) to yield pentapeptidyl-S-T(5), 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-T(5) was generated, implying that only D-L-L-D-S-T(4) was utilized, proving C(5) is indeed a (D)C(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 C(5) only transfers D-L-L-L-tetrapeptidyl-S-T(4) after in situ epimerization by the E domain. These results contrast the observation that C(5) 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-T(4) versus the natural peptidyl-S-T(4) donor produced misleading information regarding the specificity of the condensation domain.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Clugston
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Sieber
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D049148:Aminoacylation / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003065:Coenzyme A / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D019998:Racemases and Epimerases / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity; D014440:Tyrocidine / Q000096:biosynthesis* / Q000737:chemistry*
chemical_list: D004151:Dipeptides; D009842:Oligopeptides; D014440:Tyrocidine; D019998:Racemases and Epimerases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; C035180:tyrocidine synthetase; D003065:Coenzyme A