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Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Biology

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

E. Sutherland, C. J. Harding, et al.

Discover how Emmajay Sutherland, Christopher John Harding, and Clarissa Melo Czekster have engineered cyclodipeptide synthases to produce valuable histidine-containing cyclic dipeptides. This groundbreaking research not only uncovers the histidine selection mechanism but also opens avenues for creating a diverse library of cyclic dipeptide analogs using various amino acids.

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~3 min • Beginner • English
Abstract
Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.
Publisher
Communications Chemistry
Published On
May 17, 2022
Authors
Emmajay Sutherland, Christopher John Harding, Clarissa Melo Czekster
Tags
cyclodipeptide synthases
cyclic dipeptides
mutagenesis
histidine
amino acids
bioactive molecules
protein engineering
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