Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

Publikation: Beitrag in FachzeitschriftForschungsartikelBeigetragenBegutachtung

Beitragende

Abstract

BACKGROUND: Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however.

RESULTS: The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired.

CONCLUSIONS: A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.

Details

OriginalspracheEnglisch
Seiten (von - bis)287-297
Seitenumfang11
FachzeitschriftCell Chemical Biology
Jahrgang7
Ausgabenummer4
PublikationsstatusVeröffentlicht - Apr. 2000
Peer-Review-StatusJa

Externe IDs

Scopus 0034176433
ORCID /0000-0002-2331-2221/work/142242777

Schlagworte

Schlagwörter

  • Bacterial Proteins/metabolism, Binding Sites, Cloning, Molecular, Dactinomycin/analogs & derivatives, Dipeptides/biosynthesis, Gene Expression Regulation, Bacterial, Methylation, Multienzyme Complexes/genetics, Peptide Synthases/genetics, Peptides/chemical synthesis, Recombinant Proteins/genetics, Streptomyces/enzymology

Bibliotheksschlagworte