ACS Chem Biol. 2026 May 22. doi: 10.1021/acschembio.6c00013. Online ahead of print.

ABSTRACT

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.

PMID:42172238 | DOI:10.1021/acschembio.6c00013