Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides.
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.
Schauwecker
F
F
Max-Volmer-Institut, Fachgebiet Biochemie und Molekulare Biologie, Technische Universität Berlin, Berlin-Charlottenburg, 10587, Germany.
Pfennig
F
F
Grammel
N
N
Keller
U
U
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003609:Dactinomycin / Q000031:analogs & derivatives / Q000096:biosynthesis*; D004151:Dipeptides / Q000096:biosynthesis; D015964:Gene Expression Regulation, Bacterial; D008745:Methylation; D009097:Multienzyme Complexes / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics; D010455:Peptides / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology*
D001426:Bacterial Proteins; D004151:Dipeptides; D009097:Multienzyme Complexes; D010455:Peptides; D011994:Recombinant Proteins; D003609:Dactinomycin; D010453:Peptide Synthases; C052258:actinomycin synthetase II; C052259:actinomycin synthetase III