Valinophos Reveals a New Route in Microbial Phosphonate Biosynthesis That Is Broadly Conserved in Nature

Abstract

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.

Biosynthesis of Bacillus Phosphonoalamides Reveals Highly Specific Amino Acid Ligation

“…Having established the synthesis of PnAla from PEP by PnfC and PnfD, we sought to reconstitute the reactions leading to peptide formation. The remaining genes in the BGC, pnfA and pnfB , encoded putative ATP-grasp ligases, are a family of enzymes that implicated formation of other phosphonopeptides including valinophos, rhizocticin, and plumbemycin pathways. ,− In canonical peptide bond formation by ATP-grasp ligases, one amino acid is first activated as a carboxylate, forming an acylphosphate intermediate upon ATP hydrolysis. The amine of the second amino acid is then primed for nucleophilic attack, forming an amide bond.…”

“…The rhizocticin, plumbemycin, valinophos, and Streptomyces phosphonoalamide pathways all use ATP-grasp ligases to produce multiple compounds with the same Pn warhead. 7,9,19,20,28 Moreover, the composition of proteinogenic amino acids within these antimicrobial phosphonopeptides has been shown to influence their selectivity. 29,35,36 Most strikingly, the rhizocticins and plumbemycins both contain the threonine synthase inhibitor APPA as a C-terminal residue.…”

“…Outside of the Bacillus phosphonoalamides, ATP-grasp ligases appear to underlie a strategy for producing multiple phosphonopeptides from a single pathway. The rhizocticin, plumbemycin, valinophos, and Streptomyces phosphonoalamide pathways all use ATP-grasp ligases to produce multiple compounds with the same Pn warhead. ,,,, Moreover, the composition of proteinogenic amino acids within these antimicrobial phosphonopeptides has been shown to influence their selectivity. ,, Most strikingly, the rhizocticins and plumbemycins both contain the threonine synthase inhibitor APPA as a C -terminal residue. However, the rhizocticins are antifungals while the plumbemycins display antibacterial activity. − Phosphonoalamide A (PnAla–Ala–Val, from Streptomyces ) and phosphonoalamide F (Ala-Ala-PnAla, from Bacillus ) also exhibit different spectra of antimicrobial activity, , but it remains to be seen whether this is due to amino acid composition or position of the PnAla moiety.…”

“…Pn NPs have found commercial success in multiple industries, inspiring new genomics-driven techniques for their discovery . In the past decade alone, genome mining and targeted isolation have yielded multiple new Pn scaffolds, with new chemotypes, previously unknown headgroups, and a wide array of bioactivities. − However, genomic data suggests that an even greater number of Pn NPs await discovery, as nearly 7% of all bacteria are predicted to harbor biosynthetic gene clusters (BGCs) for the compounds, very few of which encode for known products . To tap into this wealth of compounds, a greater understanding of Pn biosynthesis is required to accurately predict and classify the pathways, products, and potential biological activities of these BGCs.…”

Complete Biosynthetic Pathway of the Phosphonate Phosphonothrixin: Two Distinct Thiamine Diphosphate-Dependent Enzymes Divide the Work to Form a C–C Bond

“…11 It was demonstrated very recently that four enzymes, VlpB−E, for the biosynthesis of the phosphonate valinophos function continuously to convert 3 to 2-hydroxy-3-phosphonopropanoic acid (HPPA; 4) followed by the reduction to form (2,3-dihydroxypropyl)phosphonic acid (DPPA, 5) (Figure S2). 12,13 The significant similarity of VlpB− E to the C-terminal region of PtxB10 (PtxB10C) and PtxB12− 14 strongly suggested that PtxB10C and PtxB12−14 also produce 5 from 3 (Figure S2). However, the functions of each PtxB enzyme remained to be biochemically elucidated, and the mechanism of elongation of a two-carbon unit to obtain PTX from 5 remained particularly elusive.…”