A Cysteine-Dependent Peptide Cyclase with Broad Substrate Tolerance Enables Chemoenzymatic Synthesis of Macolacin Analogs.

Thioesterase (TE) domains catalyze peptide release and macrocyclization during nonribosomal peptide biosynthesis, typically via catalytic serine. In contrast, MacB-TE from macolacin biosynthesis employs a rare cysteine nucleophile, raising questions regarding the catalytic requirements of cysteine-dependent peptide cyclases. Here, we report the biochemical and functional characterization of MacB-TE and demonstrate its efficiency, regioselectivity, and exclusive macrocyclization. Using a synthetic thioester substrate, MacB-TE converted >99% of the linear precursor into the cyclic product within minutes. Mutational analysis demonstrated that Cys89 is indispensable for ring formation, whereas His218 and Asp116 support an efficient turnover. Notably, a D116S variant retained substantial cyclization activity, uncovering an unexpected tolerance at the acidic position of the catalytic triad. Comprehensive substrate profiling showed that MacB-TE accommodated extensive side-chain variations and diverse N-terminal lipid groups. The enzyme efficiently macrocyclized all nine alanine-scan variants, with six positions supporting near-quantitative conversion and the remaining three producing more than 80% of the cyclic products. Likewise, lipid-modified substrates, including those with aliphatic and aromatic motifs, were quantitatively converted into their corresponding macrocycles, indicating that lipid variation had a negligible impact on catalysis. Collectively, these findings establish MacB-TE as a highly efficient and broadly tolerant macrocyclase, enabling the chemoenzymatic synthesis of macolacin analogs and expanding the mechanistic understanding of Cys-type TEs.