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bioRxiv · 10.1101/2020.05.12.092031

Promiscuous enzymes cooperate at the substrate level en route to lactazole A

Vinogradov, A. A.·Shimomura, M.·Kano, N.·Goto, Y.·Onaka, H.·Suga, H.
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Abstract

Enzymes involved in ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis often have relaxed specificity profiles and are able to modify diverse substrates. When several such enzymes act together during precursor peptide maturation, a multitude of products can form, and yet usually, the biosynthesis converges on a single natural product. For the most part, the mechanisms controlling the integrity of RiPP assembly remain elusive. Here, we investigate biosynthesis of lactazole A, a model thiopeptide produced by five promiscuous enzymes from a ribosomal precursor peptide. Using our in vitro thiopeptide production (FIT-Laz) system, we determine the order of biosynthetic events at the individual modification level, and supplement this study with substrate scope analysis for participating enzymes. Combined, our results reveal a dynamic thiopeptide assembly process with multiple points of kinetic control, intertwined enzymatic action, and the overall substrate-level cooperation between the enzymes. This work advances our understanding of RiPP biosynthesis processes and facilitates thiopeptide bioengineering.

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Vinogradov, A. A., Shimomura, M., Kano, N., Goto, Y., Onaka, H., Suga, H.. 2020-05-14. Promiscuous enzymes cooperate at the substrate level en route to lactazole A. https://doi.org/10.1101/2020.05.12.092031

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