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Phosphonate natural products have a history of successful application in medicine and biotechnology due to their ability to inhibit essential cellular pathways. This has inspired efforts to discover phosphonate natural products by prioritizing microbial strains whose genomes encode uncharacterized biosynthetic gene clusters (BGCs). Thus, success in genome mining is dependent on establishing the fundamental principles underlying the biosynthesis of inhibitory chemical moieties to facilitate accurate prediction of BGCs and the bioactivities of their products. Here, we report the complete biosynthetic pathway for the argolaphos phosphonopeptides. We uncovered the biochemical origins of aminomethylphosphonate (AMPn) and N ε -hydroxyarginine, two noncanonical amino acids integral to the antimicrobial function of argolaphos. Critical to this pathway were dehydrogenase and transaminase enzymes dedicated to the conversion of hydroxymethylphosphonate to AMPn. The interconnected activities of both enzymes provided a solution to overcome unfavorable energetics, empower cofactor regeneration, and mediate intermediate toxicity during these transformations. Sequential ligation of l-arginine and l-valine was afforded by two GCN5-related N-acetyltransferases in a tRNA-dependent manner. AglA was revealed to be an unusual heme-dependent monooxygenase that hydroxylated the N ε position of AMPn-Arg. As the first biochemically characterized member of the YqcI/YcgG protein family, AglA enlightens the potential functions of this elusive group, which remains biochemically distinct from the well-established P450 monooxygenases. The widespread distribution of AMPn and YqcI/YcgG genes among actinobacterial genomes suggests their involvement in diverse metabolic pathways and cellular functions. Our findings illuminate new paradigms in natural product biosynthesis and realize a significant trove of AmPn and N ε -hydroxyarginine natural products that await discovery.

Phosphonate natural products are potent inhibitors of cellular metabolism with an established record of commercialization in medicine and biotechnology. Although genome mining has emerged as an accelerated method for the discovery of new phosphonates, a robust framework of their metabolism is needed to identify the pathways most likely to yield compounds with desired activities. Here we expand our understanding of these natural products by reporting the complete biosynthetic pathway for valinophos, a phosphonopeptide natural product containing the unusual ( R )-2,3-dihydroxypropylphosphonate (DHPPA) scaffold. The pathway was defined by several enzymatic transformations and intermediates previously unknown to phosphonate natural products. A dedicated dehydrogenase served as a new phosphoenolpyruvate mutase coupling enzyme. Notably, its reduction of phosphonopyruvate to phosphonolactate defined a new early branchpoint in phosphonate biosynthesis. Functionally interconnected kinase and reductase enzymes catalyzed reactions reminiscent of glycolysis and arginine biosynthesis to produce a transient, but essential, phosphonolactaldehyde intermediate. We demonstrate esterification of l-valine onto DHPPA as a new biochemical activity for ATP-Grasp ligase enzymes. Unexpectedly, a second amino acid ligase then adjoined additional amino acids at the valinyl moiety to produce a suite of DHPPA-dipeptides. The genes for DHPPA biosynthesis were discovered among genomes of bacteria from wide-ranging habitats, suggesting a wealth of unknown compounds that may originate from this core pathway. Our findings establish new biosynthetic principles for natural products and provide definition to unexplored avenues for bioactive phosphonate genome mining.