Biosynthesis of fosfazinomycin is a convergent process

Abstract

Fosfazinomycin A is a phosphonate natural product in which the C-terminal carboxylate of a Val-Arg dipeptide is connected to methyl 2-hydroxy-2-phosphono-acetate (Me-HPnA) via a unique hydrazide linkage. We report here that Me-HPnA is generated from phosphonoacetaldehyde (PnAA) in three biosynthetic steps through the combined action of an O-methyltransferase (FzmB) and an α-ketoglutarate (α-KG) dependent non-heme iron dioxygenase (FzmG). Unexpectedly, the latter enzyme is involved in two different steps, oxidation of the PnAA to phosphonoacetic acid as well as hydroxylation of methyl 2-phosphonoacetate. The N-methyltransferase (FzmH) was able to methylate Arg-NHNH₂ (3) to give Arg-NHNHMe (4), constituting the second segment of the fosfazinomycin molecule. Methylation of other putative intermediates such as desmethyl fosfazinomycin B was not observed. Collectively, our current data support a convergent biosynthetic pathway to fosfazinomycin.

Fig. 1. Structures of select phosphonates.

Fig. 2. Three disconnections to generate the central hydrazide core of fosfazinomycins.

Scheme 1. Previously proposed initial steps of the biosynthetic pathway of fosfazinomycin A.

Fig. 3. ³¹P NMR spectra of the products obtained by incubation of His₆–FzmG with Me-PnA. (A) NMR spectrum of the reaction mixture containing Me-PnA, O₂, α-KG, Fe(ii), l-ascorbate, and His₆–FzmG. (B) NMR spectrum of the enzymatic reaction mixture spiked with authentic standard of Me-HPnA. (C) NMR spectrum of the reaction mixture in the absence of α-KG. (D) NMR spectrum of the reaction mixture in the absence of l-ascorbate. (E) NMR spectrum of the reaction mixture in the absence of His₆–FzmG. (F) NMR spectrum of authentic Me-HPnA.

Fig. 4. ³¹P NMR spectra of the incubation of His₆–FzmG with PnAA. (A) NMR spectrum of the reaction mixture containing PnAA, O₂, α-KG, Fe(ii), l-ascorbate, and His₆–FzmG. (B) NMR spectrum of the enzymatic reaction mixture spiked with authentic standard of PnAA. (C) NMR spectrum of the reaction mixture spiked with authentic standards of PnAA and PnA. (D) NMR spectrum of the reaction mixture in the absence of His₆–FzmG. The chemical shifts of phosphonates in ³¹P NMR are very sensitive to pH near their pK_a values, accounting for the changes in chemical shifts between experiments and requiring spiking with standards for assignments.

Scheme 2. Two proposed routes of converting (S)-Me-HPnA to fosB.

Fig. 5. ¹H NMR spectra of products generated upon incubation of His₆–FzmH with Arg-NHNH₂ (3). (A) NMR spectrum of the reaction mixture in the absence of His₆–FzmH. (B) NMR spectrum of the enzymatic reaction mixture containing Arg-NHNH₂, SAM, AdoHcy nucleosidase, and His₆–FzmH. (C) NMR spectrum of the reaction mixture spiked with authentic standard of Arg-NHNHMe. The peaks marked with asterisks originate from SAM and SAM-related products.