Peptide Heterocyclization: The Defining Modification for an Emerging Natural Product Class

The thiazole/oxazole-modified microcins (TOMMs) are a recently grouped class of ribosomally synthesized and posttranslationally modified peptides. Encoded by many bacteria and archaea, these natural products are structurally and functionally diverse. Previous work has demonstrated that an evolutionarily conserved, heterotrimeric synthetase is responsible for azole biogenesis. This ‘TOMM’ synthetase transforms select serines, threonines and cysteines into azole heterocycles via a tandem cyclodehydration-dehydrogenation reaction. Although studied by a number of groups since the 1990’s, the exact roles of each component of the TOMM synthetase and the mechanism of azoline formation remained poorly understood. Using a novel TOMM synthetase from Bacillus sp. Al Hakam, it was discovered that cyclodehydration proceeds via the direct, ATP-dependent phosphorylation of the amide backbone carbonyl oxygen. Moreover, a detailed dissection of the synthetase complex demonstrated that this modification is performed by the YcaO homolog (D-protein) that exists in all TOMM biosynthetic gene clusters, providing the first function for a member the enigmatic superfamily. An X-ray structure of a YcaO facilitated the identification of a novel ATP-binding fold and motif. Finally, through a combination of biophysical experiments and the use of site-directed mutagenesis, the role of the MoeB/ThiF homolog (C-protein) in the cluster has also been addressed. Together, these studies provide a foundation for a deeper understanding of how azole biogenesis occurs and, more broadly, insight into the functions of YcaO proteins not associated with TOMM biosynthetic clusters.