Biosynthesis of Bacillus Phosphonoalamides Reveals Highly Specific Amino Acid Ligation.

Phosphonate natural products have a history of commercial success across numerous industries due to their potent inhibition of metabolic processes. Over the past decade, genome mining approaches have successfully led to the discovery of numerous bioactive phosphonates. However, continued success is dependent upon a greater understanding of phosphonate metabolism, which will enable the prioritization and prediction of biosynthetic gene clusters for targeted isolation. Here, we report the complete biosynthetic pathway for phosphonoalamides E and F, antimicrobial phosphonopeptides with a conserved C-terminal l-phosphonoalanine (PnAla) residue. These peptides, produced by Bacillus, are the direct result of PnAla biosynthesis and serial ligation by two ATP-grasp ligases. A critical step of this pathway was the reversible transamination of phosphonopyruvate to PnAla by a dedicated transaminase with preference for the forward reaction. The dipeptide ligase PnfA was shown to ligate alanine to PnAla to afford phosphonoalamide E, which was subsequently ligated to alanine by PnfB to form phosphonoalamide F. Specificity profiling of both ligases found each to be highly specific, although the limited acceptance of noncanonical substrates by PnfA allowed for in vitro formation of products incorporating alternative pharmacophores. Our findings further establish the transaminative branch of phosphonate metabolism, unveil insights into the specificity of ATP-grasp ligation, and highlight the biocatalytic potential of biosynthetic enzymes.

Phosphonoalamides Reveal the Biosynthetic Origin of Phosphonoalanine Natural Products and a Convergent Pathway for Their Diversification.

Phosphonate natural products, with their potent inhibitory activity, have found widespread use across multiple industries. Their success has inspired development of genome mining approaches that continue to reveal previously unknown bioactive scaffolds and biosynthetic insights. However, a greater understanding of phosphonate metabolism is required to enable prediction of compounds and their bioactivities from sequence information alone. Here, we expand our knowledge of this natural product class by reporting the complete biosynthesis of the phosphonoalamides, antimicrobial tripeptides with a conserved N-terminal l-phosphonoalanine (PnAla) residue produced by Streptomyces. The phosphonoalamides result from the convergence of PnAla biosynthesis and peptide ligation pathways. We elucidate the biochemistry underlying the transamination of phosphonopyruvate to PnAla, a new early branchpoint in phosphonate biosynthesis catalyzed by an aminotransferase with evolved specificity for phosphonate metabolism. Peptide formation is catalyzed by two ATP-grasp ligases, the first of which produces dipeptides, and a second which ligates dipeptides to PnAla to produce phosphonoalamides. Substrate specificity profiling revealed a dramatic expansion of dipeptide and tripeptide products, while finding PnaC to be the most promiscuous dipeptide ligase reported thus far. Our findings highlight previously unknown transformations in natural product biosynthesis, promising enzyme biocatalysts, and unveil insights into the diversity of phosphonopeptide natural products.