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.

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.