The Biosynthetic Pathway to the Pyrroloiminoquinone Marine Natural Product Ammosamide C

Ammosamide C is a marine natural product containing a highly decorated pyrroloiminoquinone core. Studies on the biosynthetic gene cluster (BGC) that produces ammosamides previously revealed that they are made by a series of posttranslational modifications (PTMs). The BGC includes genes encoding a precursor peptide AmmA and four enzymes known as PEptide Aminoacyl-tRNA Ligases (PEARLs). Initial studies into the ammosamide biosynthetic pathway demonstrated Trp addition to a precursor peptide by the PEARL AmmB₂. Thereafter, sequential modifications by several enzymes, including two other PEARLs lead to the formation of a peptide intermediate bearing a C-terminal diaminoquinone. In the present work, we present the biosynthetic steps that convert this intermediate to ammosamide C. The PEARL AmmB₄ unexpectedly appends an arginine to the C-terminus of the aforementioned intermediate. Then, C-terminal proteolysis by the heterodimeric TldD/E-like protease Amm12/13 releases a dipeptide, which is subsequently cleaved by the dipeptidase Amm19 to produce a Trp-derived diaminoquinone. Amm3 next catalyzes the conversion of this Trp derivative to the corresponding chlorinated ammosamaic acid. Finally, a putative aminotransferase Amm20 performs an amidation, and Amm23 methylates this intermediate to arrive at ammosamide C; the order of these last two steps could not be determined definitively. This study reveals an unexpectedly lengthy route to ammosamide that illustrates the opportunistic nature of natural product biosynthesis, demonstrates a role for a PEARL that is unlike previous roles, identifies steps that are not PTMs, and adds Arg-tRNA to the growing repertoire of aminoacyl tRNAs that are used by PEARLs.