Non-Ribosomal Peptide Synthetases (NRPSs) assemble a diverse range of natural products with important applications in both medicine and agriculture. They consist of several multienzyme subunits that must interact with each other in a highly controlled manner to facilitate efficient chain transfer, thus ensuring biosynthetic fidelity. Several mechanisms for chain transfer are known for NRPSs, promoting structural diversity. Herein, we report the first biochemically characterized example of a type II thioesterase (TEII) domain capable of catalysing aminoacyl chain transfer between thiolation (T) domains on two separate NRPS subunits responsible for installation of a dehydrobutyrine moiety. Biochemical dissection of this process reveals the central role of the TEII-catalysed chain translocation event and expands the enzymatic scope of TEII domains beyond canonical (amino)acyl chain hydrolysis. The apparent co-evolution of the TEII domain with the NRPS subunits highlights a unique feature of this enzymatic cassette, which will undoubtedly find utility in biosynthetic engineering efforts.
Bibliographical noteFunding Information:
This work was supported by the Biotechnology and Biological Sciences Research Council UK (S.W. and H.D., BB/P00380X/1 and BB/R00479X/1, W.B. and S.L.C., BB/P003656/1). S.W and H.D. are grateful for SFC Covid-19 Grant extension and bridging Fund and UKRI Covid-19 Extension Allocation Fund. Q.Z. and Y.Y. are grateful for financial support from the National Key Research and Development Program of China (2018YFA0900400), the National Natural Science Foundation of China (31570033, 31811530299, and 31870035 to Y.Y.). H.D. and Y.Y. are recipients of a Royal Society-NSFC Newton Mobility Grant Award (IEC\NSFC\170617). M.J. is the recipient of a BBSRC Discovery Fellowship (BB/R012121/1). The Bruker MaXis II instrument used in this study was funded by the BBSRC (BB/M017982/1). K.K. and H.D. thank Leverhulme Trust-Royal Society Africa award (AA090088) and the jointly funded UK Medical Research Council-UK Department for International Development (MRC/DFID) Concordat agreement African Research Leaders Award (MR/S00520X/1).
© 2022, The Author(s).
- Enzyme mechanisms