Use of a Phosphonate Methyltransferase in the Identification of the Fosfazinomycin Biosynthetic Gene Cluster

Abstract

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for “stable isotope labeling of phosphonates in extract” (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S‐adenosyl methionine. Mass‐guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

“…Intriguingly, all but one ( lom30 / SAMT0149 ) of the kin / lom -type diazo-forming enzymes are encoded within the gene cluster that produces the hydrazide-containing natural product fosfazinomycin ( fzm ), further supporting their role in N–N bond formation (Scheme 19). 430 The relationship between the lom / kin -type diazo-forming gene cassette and the fzm -type hydrazide-forming gene cassette will be discussed below in the section covering fosfazinomycin biosynthesis (Section 3.3.1. ).…”

“…WM6372 and the identification of the fosfazinomycin ( fzm ) biosynthetic gene cluster using genome mining and a novel enzymatic isotope labeling approach termed SILPE (stable isotope labeling of phosphonates in extracts). 430 Interestingly, the fzm gene cluster contains putative diazo-forming gene cassettes from both the cremeomycin and kinamycin/lomaiviticin pathway. In vitro characterization of enzymes from both of these gene cassettes has led to proposals for their roles in hydrazide biosynthesis.…”

Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways

“…Most recently we have developed a stable isotope strategy to discover novel phosphonate compounds by reacting partially purified culture extracts with phosphonate O-methyltransferase DhpI (from dehydrophos biosynthesis) [35] and a mixture of SAM and CD 3 -SAM [17]. The chemoselective activity of DhpI enables identification by searching for pairs of compounds with an exact isotopic difference of 3.0188 Da, corresponding to SAM- and CD 3 -SAM-methylated phosphonates.…”

The many roles of glutamate in metabolism

“…Analysis of genomes revealed a very common occurrence of a pair of GS and amide hydrolase or cyclotransferase genes in gene clusters [32]. One intriguing example is in a set of five genes that appear to be conserved in clusters of natural products containing N-N bonds such as the hydrazine-containing fosfazinomycin [24] and the diazo-containing lomaiviticin [35]. Thus, it is possible that glutamate may be in some way involved in the process of N-N bond formation.…”