Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3

Abstract

Fom3, the antepenultimate enzyme in the fosfomycin biosynthetic pathway in Streptomyces spp., is a class B cobalamin-dependent radical SAM methyltransferase that catalyzes methylation of (5'-cytidylyl)-2-hydroxyethylphosphonate (2-HEP-CMP) to form (5'-cytidylyl)-2-hydroxypropylphosphonate (2-HPP-CMP). Previously, the reaction of Fom3 with 2-HEP-CMP produced 2-HPP-CMP with mixed stereochemistry at C2. Mechanistic characterization has been challenging because of insoluble expression and poor cobalamin (B₁₂) incorporation in Escherichia coli. Recently, soluble E. coli expression and incorporation of cobalamin into Fom3 were achieved by overexpression of the BtuCEDFB cobalamin uptake system. Herein, we use this new method to obtain Fom3 from Streptomyces wedmorensis. We show that the initiator 5'-deoxyadenosyl radical stereospecifically abstracts the pro- R hydrogen atom from the C2 position of 2-HEP-CMP and use the downstream enzymes FomD and Fom4 to demonstrate that our preparation of Fom3 produces only (2 S)-2-HPP-CMP. Additionally, we show that the added methyl group originates from SAM under multiple-turnover conditions, but the first turnover uses a methyl donor already present on the enzyme; furthermore, cobalamin isolated from Fom3 reaction mixtures contains methyl groups derived from SAM. These results are consistent with a model in which Fom3 catalyzes methyl transfer from SAM to cobalamin and the resulting methylcobalamin (MeCbl) is the ultimate methyl source for the reaction.

Figure 1

(a) Liquid chromatography–mass spectrometry (LC–MS) analysis of 2-HEP-CMP (black), [2-²H₂]-2-HEP-CMP (red), (2R)-[2-²H₁]-2-HEP-CMP (green), and (2S)-[2-²H₁]-2-HEP-CMP (blue). (b) LC–MS analysis of 2-HPP-CMP (left) and 5′-dA (right) produced from SUMO-Fom3 reactions with 2-HEP-CMP isotopologues.

Figure 2

Stereochemistry determination of the Fom3 product. (a) Methyl signals in ¹H NMR spectra (D₂O, 600 MHz) of standards of (2R)-2-HPP-CMP, (2S)-2-HPP-CMP, and a mixture of (2R)- and (2S)-2-HPP-CMP. (b) ³¹P NMR spectra (D₂O, 600 MHz) of the products after reaction of FomD and Fom4 with a mixture of (2R)- and (2S)-2-HPP-CMP standards (top) and with 2-HPP-CMP produced by Fom3 (bottom), demonstrating that only fosfomycin is formed from the Fom3 product and not 2-OPP.

Figure 3

LC–MS analysis of 2-HPP-CMP from SUMO-Fom3 reactions with CD₃-SAM and/or CD₃Cbl under (a) multiple-turnover conditions (20 μM SUMO-Fom3 and 1 mM 2-HEP-CMP) and (b) single-turnover conditions (100 μM SUMO-Fom3 and 50 μM 2-HEP-CMP). Formation of the unlabeled product in single-turnover reactions with CD₃-SAM and no added MeCbl demonstrates that as-isolated SUMO-Fom3 contains transferable methyl groups.

Scheme 1

Biosynthesis of Fosfomycin in Streptomyces spp.

Scheme 2

Differentiation of (2R)- and (2S)-2-HPP-CMP

Scheme 3

Proposed Mechanism for Fom3