TY - JOUR
T1 - Unimodular Methylation by Adenylation–Thiolation Domains Containing an Embedded Methyltransferase
AU - Mori, Shogo
AU - Garneau-Tsodikova, Sylvie
AU - Tsodikov, Oleg V.
N1 - Funding Information:
This work was supported by a National Science Foundation (NSF) CAREER Award MCB-1149427 (to S.G.-T.) and by startup funds from the University of Kentucky College of Pharmacy (to S.G.-T. and O.V.T.). Note: The authors declare no competing financial interest.
Publisher Copyright:
© 2020 Elsevier Ltd
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/10/2
Y1 - 2020/10/2
N2 - Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.
AB - Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.
KW - biosynthesis
KW - enzyme mechanism
KW - interrupted adenylation domain
KW - natural product
KW - nonribosomal peptide synthetase
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U2 - 10.1016/j.jmb.2020.09.004
DO - 10.1016/j.jmb.2020.09.004
M3 - Article
C2 - 32920052
AN - SCOPUS:85091497427
VL - 432
SP - 5802
EP - 5808
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 21
ER -