# Exploring the Substrate Flexibility of GrsB Thioesterase Leads to the Structural Reassignment of a Gramicidin S Variant

## TL;DR

Researchers found that a thioesterase enzyme can process modified building blocks to make a variant of gramicidin S, leading to a structural correction of the variant.

## Contribution

The study reveals stereochemical flexibility in GrsB-TE and corrects the structure of a reported GS variant.

## Key findings

- GrsB-TE can cyclize a substrate with L-Ser(Allyl) at position 6 but not at position 1.
- The reported GS-variant actually contains D-Ser(Allyl) instead of L-Ser(Allyl).
- Structural and functional analyses confirmed the stereochemical flexibility of GrsB-TE.

## Abstract

Gramicidin S (GS) is a cyclic decapeptide derived from two pentapeptides. The C‐terminal thioesterase (TE) domain of gramicidin S synthetase B (GrsB) dimerizes precursor pentapeptides and cyclizes the resulting linear decapeptide. Recently, a GS variant (GS‐SA), in which a single D‐Phe is replaced by L‐Ser(Allyl), is reported via precursor‐directed biosynthesis in a native GS producer. To understand how GrsB‐TE processes such modified precursors, its substrate specificity using synthetic linear peptides is investigated. GrsB‐TE cyclizes a substrate containing L‐Ser(Allyl) at position 6 but not at position 1. However, the enzymatically synthesized GS‐SA shows a different high‐performance liquid chromatography retention time than that of the reported GS variant. Further structural and functional analyses, including 1H nuclear magnetic resonance, antimicrobial assays, and circular dichroism spectroscopy, reveal that the reported GS‐SA contained D‐Ser(Allyl) rather than L‐Ser(Allyl). These findings reveal a previously unrecognized stereochemical flexibility in GrsB‐TE and support the structural revision of the reported GS variant.

Investigation of the substrate specificity of gramicidin S synthetase B‐thioesterase reveals stereochemical flexibility, enabling the production of gramicidin S (GS) analogs with L‐ or D‐Ser(Allyl). Structural reassignment of a GS variant (GS‐SA) obtained from precursor‐directed biosynthesis is achieved using liquid chromatography‐mass spectrometry, 1H nuclear magnetic resonance, and antimicrobial assays.© 2025 WILEY‐VCH GmbH