Journal
BIOCHEMISTRY
Volume 42, Issue 41, Pages 12095-12104Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi035090+
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- NIGMS NIH HHS [GM 20011] Funding Source: Medline
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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 (C-D(L)). To validate this prediction and establish that the C-5 domain of tyrocidine synthetase is indeed C-D(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 CL 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.
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