The Biosynthetic Pathway to the Pyrroloiminoquinone Marine Natural Product Ammosamide C

Abstract

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, Amm23 methylates this intermediate and a putative aminotransferase Amm20 performs an amidation to arrive at ammosamide C; the order of these last two steps could not be determined. 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 amino acyl tRNAs that are used by PEARLs.