Unexpected Transformations during Pyrroloiminoquinone Biosynthesis

Abstract

Pyrroloiminoquinone-containing natural products have long been known for their biological activities. They are derived from tryptophan, but their biosynthetic pathways have remained elusive. Studies on the biosynthetic gene cluster (BGC) that produces the ammosamides revealed that the first step is attachment of Trp to the C-terminus of a scaffold peptide in an ATP- and tRNA-dependent manner catalyzed by a PEptide Aminoacyl-tRNA Ligase (PEARL). The indole of Trp is then oxidized to a hydroxyquinone. We previously proposed a chemically plausible and streamlined pathway for converting this intermediate to the ammosamides using additional enzymes encoded in the BGC. In this study, we report the activity of four additional enzymes from two gene clusters, which show that the previously proposed pathway is incorrect and that Nature’s route toward pyrroloiminoquinones is much more complicated. We demonstrate that, surprisingly, amino groups in pyrroloiminoquinones are derived from (at least) three different sources, glycine, asparagine, and leucine, all introduced in a tRNA-dependent manner. We also show that an FAD-dependent putative glycine oxidase (Amm14) is required for the process that incorporates the nitrogens from glycine and leucine and that a quinone reductase is required for the incorporation of asparagine. Additionally, we provide the first insights into the evolutionary origin of the PEARLs as well as related enzymes, such as the glutamyl-tRNA-dependent dehydratases involved in the biosynthesis of lanthipeptides and thiopeptides. These enzymes appear to all have descended from the ATP-GRASP protein family.

PEARL-Catalyzed Peptide Bond Formation after Chain Reversal by Ureido-Forming Condensation Domains

“…PEARLs and the glutamylation domains in class I lantibiotic dehydratases use aminoacyl-tRNA as substrates and share conserved residues that are proposed to bind the 5′-phosphate of the aminoacyl-tRNA , (Figure S12). PEARLs possess additional conserved residues (Figure S12) for the ATP-dependent phosphorylation activity .…”

“…PEARLs and the glutamylation domains in class I lantibiotic dehydratases use aminoacyl-tRNA as substrates and share conserved residues that are proposed to bind the 5′-phosphate of the aminoacyl-tRNA , (Figure S12). PEARLs possess additional conserved residues (Figure S12) for the ATP-dependent phosphorylation activity . Since PEARLs are not involved in dehydroamino acid synthesis, they do not co-occur with the elimination domains (PF14028) of lantibiotic dehydratases in the genome neighborhood.…”

“…PEARLs possess additional conserved residues ( Figure S12 ) for the ATP-dependent phosphorylation activity. 30 Since PEARLs are not involved in dehydroamino acid synthesis, they do not co-occur with the elimination domains (PF14028) of lantibiotic dehydratases in the genome neighborhood. Therefore, sequence analysis and genome neighborhood analysis can distinguish PEARLs from lantibiotic dehydratases.…”

“…They generate the dehydroamino acids of ribosomally synthesized and post-translationally modified peptides (RiPPs), including lanthipeptides (called lantibiotics if they display antibiotic activity) and thiopeptides . The dehydration reaction involves the glutamylation of serine and threonine hydroxyl groups using glutamyl-tRNA Glu catalyzed by the N-terminal domain of the enzyme, and subsequent elimination of glutamate by the C-terminal domain to generate peptidyl dehydroamino acids (Figure C ). , Enzymes frequently mis-annotated as lantibiotic dehydratases are peptide aminoacyl-tRNA ligases (PEARLs). − PEARLs share sequence homology with the glutamylation domain of lantibiotic dehydratases, but catalyze peptide bond formation at the C-terminus of a carrier peptide using adenosine-5′-triphosphate (ATP) and aminoacyl-tRNA (Figure C ) . The amino acid added by the PEARL typically undergoes further enzymatic modifications and proteolysis to yield an amino acid-derived natural product. − However, genes for a RiPP precursor peptide or a cognate PEARL carrier peptide cannot be identified in the NRPS BGCs of interest in this study, indicating the putative lantibiotic dehydratases serve a different function in the biosynthesis of NRPs.…”

“… 21 , 26 Enzymes frequently mis-annotated as lantibiotic dehydratases are peptide aminoacyl-tRNA ligases (PEARLs). 27 − 30 PEARLs share sequence homology with the glutamylation domain of lantibiotic dehydratases, but catalyze peptide bond formation at the C-terminus of a carrier peptide using adenosine-5′-triphosphate (ATP) and aminoacyl-tRNA ( Figure 1 C ). 28 The amino acid added by the PEARL typically undergoes further enzymatic modifications and proteolysis to yield an amino acid-derived natural product.…”