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

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 was replaced by L-Ser(Allyl), was reported via precursor-directed biosynthesis in a native GS producer. To understand how GrsB-TE processes such modified precursors, we investigated its substrate specificity using synthetic linear peptides. GrsB-TE cyclized a substrate containing L-Ser(Allyl) at position 6 but not at position 1. However, the enzymatically synthesized GS-SA showed a different HPLC retention time than that of the reported GS variant. Further structural and functional analyses, including 1H NMR, antimicrobial assays, and circular dichroism spectroscopy, revealed 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.