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.