TY - JOUR
T1 - Mechanistic Analysis of the Biosynthesis of the Aspartimidylated Graspetide Amycolimiditide
AU - Choi, Brian
AU - Elashal, Hader E.
AU - Cao, Li
AU - Link, A. James
N1 - Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
PY - 2022/11/30
Y1 - 2022/11/30
N2 - Several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs) are composed of multiple macrocycles. The enzymes that assemble these macrocycles must surmount the challenge of installing a single specific set of linkages out of dozens of distinct possibilities. One class of RiPPs that includes multiple macrocycles are the graspetides, named after the ATP-grasp enzymes that install ester or amide linkages between pairs of nucleophilic and electrophilic side chains. Here, using heterologous expression and NMR spectroscopy, we characterize the connectivity and structure of amycolimiditide, a 29 aa graspetide with a stem-loop structure. The stem includes four esters and extends over 20 Å. The loop of amycolimiditide is distinguished by the presence of an aspartimide moiety, installed by a dedicated O-methyltransferase enzyme. We further characterize the biosynthesis of amycolimiditide in vitro, showing that the amycolimiditide ATP-grasp enzyme AmdB operates in a strict vectorial manner, installing esters starting at the loop and proceeding down the stem. Surprisingly, the O-methyltransferase AmdM that aspartimidylates amycolimiditide prefers a substrate with all four esters installed, despite the fact that the most distal ester is ∼30 Å away from the site of aspartimidylation. This study provides insights into the structure and diversity of aspartimidylated graspetides and also provides fresh insights into how RiPP biosynthetic enzymes engage their peptide substrates.
AB - Several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs) are composed of multiple macrocycles. The enzymes that assemble these macrocycles must surmount the challenge of installing a single specific set of linkages out of dozens of distinct possibilities. One class of RiPPs that includes multiple macrocycles are the graspetides, named after the ATP-grasp enzymes that install ester or amide linkages between pairs of nucleophilic and electrophilic side chains. Here, using heterologous expression and NMR spectroscopy, we characterize the connectivity and structure of amycolimiditide, a 29 aa graspetide with a stem-loop structure. The stem includes four esters and extends over 20 Å. The loop of amycolimiditide is distinguished by the presence of an aspartimide moiety, installed by a dedicated O-methyltransferase enzyme. We further characterize the biosynthesis of amycolimiditide in vitro, showing that the amycolimiditide ATP-grasp enzyme AmdB operates in a strict vectorial manner, installing esters starting at the loop and proceeding down the stem. Surprisingly, the O-methyltransferase AmdM that aspartimidylates amycolimiditide prefers a substrate with all four esters installed, despite the fact that the most distal ester is ∼30 Å away from the site of aspartimidylation. This study provides insights into the structure and diversity of aspartimidylated graspetides and also provides fresh insights into how RiPP biosynthetic enzymes engage their peptide substrates.
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U2 - 10.1021/jacs.2c09004
DO - 10.1021/jacs.2c09004
M3 - Article
C2 - 36394830
AN - SCOPUS:85142453462
SN - 0002-7863
VL - 144
SP - 21628
EP - 21639
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 47
ER -