TY - JOUR
T1 - Characterization of a unique interrupted adenylation domain that can catalyze three reactions
AU - Lundy, Taylor A.
AU - Mori, Shogo
AU - Thamban Chandrika, Nishad
AU - Garneau-Tsodikova, Sylvie
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.). T.A.L. was in part supported by a 2018-2019 and a 2019-2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship from the College of Pharmacy at the University of Kentucky as well as a 2019-2020 Predoctoral Fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education (AFPE). S.M. was in part supported by a 2018 long-term visit fellowship from the Yamada Science Foundation, Japan. We thank A. Garzan for providing the N-Me- l -Cys and N, S-diMe- l -Cys, for which the syntheses were previously published in ref . We thank the UK PharmNMR Center (in the College of Pharmacy) for NMR support. We also thank S. Kinison and J. Horn for their help overseeing the MS/MS analysis conducted by T.A.L.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2020/1/17
Y1 - 2020/1/17
N2 - Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.
AB - Interrupted adenylation (A) domains contain auxiliary domains within their structure and are a subject of growing interest in the field of nonribosomal peptide biosynthesis. They have been shown to possess intriguing functions and structure as well as promising engineering potential. Here, we present the characterization of an unprecedented type of interrupted A domain from the columbamides biosynthetic pathway, ColG(AMsMbA). This interrupted A domain contains two back-to-back methylation (M) domains within the same interruption site in the A domain, whereas previously, naturally occurring reported and characterized interrupted A domains harbored only one M domain. By a series of radiometric and mass spectrometry assays, we show that the first and second M domains site specifically methylate the side-chain oxygen and backbone nitrogen of l-Ser after the substrate is transferred onto a carrier thiolation domain, ColG(T). This is the first reported characterization of a dimethylating back-to-back interrupted A domain. The insights gained by this work lay the foundation for future combinatorial biosynthesis of site specifically methylated nonribosomal peptides.
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U2 - 10.1021/acschembio.9b00929
DO - 10.1021/acschembio.9b00929
M3 - Article
C2 - 31887013
AN - SCOPUS:85078402257
SN - 1554-8929
VL - 15
SP - 282
EP - 289
JO - ACS Chemical Biology
JF - ACS Chemical Biology
IS - 1
ER -