Fosfomycin Biosynthesis via Transient Cytidylylation of 2-Hydroxyethylphosphonate by the Bifunctional Fom1 Enzyme

Abstract

Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, etc. Its biosynthesis starts with the formation of a carbon-phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 N-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 C-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism via such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family.

A (Re)Discovery of the Fom3 Substrate

“…Recent studies by Cho et al revealed that Fom1 from Streptomyces wedmorensis is bifunctional, containing a cytidyltransferase domain in addition to its PEP mutase domain, and that it converts HEP to cytidylyl-2-hydroxyethylphosphonate (HEP-CMP). 4 Further, a recent report in Biochemistry by Sato et al, which is the focus of this Viewpoint, revealed HEP-CMP to be the true substrate for Fom3. 3 After methylation of HEP-CMP to generate cytidylyl-2-hydroxypropylphosphonate (HPP-CMP), it is hypothesized, but not yet confirmed, that FomD, whose function was previously unknown, hydrolyzes HPP-CMP to generate ( S )-HPP, which is then converted to fosfomycin by the nonheme-iron peroxidase Fom4 (Scheme 1).…”

“…3 After methylation of HEP-CMP to generate cytidylyl-2-hydroxypropylphosphonate (HPP-CMP), it is hypothesized, but not yet confirmed, that FomD, whose function was previously unknown, hydrolyzes HPP-CMP to generate ( S )-HPP, which is then converted to fosfomycin by the nonheme-iron peroxidase Fom4 (Scheme 1). 4 …”

The intriguing biology and chemistry of fosfomycin: the only marketed phosphonate antibiotic

“…The biosynthesis of fosfomycin in Streptomyces consists of seven chemical steps. 63,64 Heterologous expression and genetic inactivation experiments indicated that the gene cluster is composed of fom1-4 and fomA-D. 65 Most of these steps (Fig. 5) have been reconstituted and characterized in vitro.…”

“…65,66 However, Fom1 is a bifunctional PepM which is CyTase-fused and identied only in the gene cluster of fosfomycin (1a) from Streptomyces strains. 63 8.1.2 The conversion of 2-HEP to HEP-CMP. This step was not proposed and conrmed until the cytidylyltransferase (CyTase) domain in Fom1 was characterized.…”

“…This step was not proposed and conrmed until the cytidylyltransferase (CyTase) domain in Fom1 was characterized. 63,64 Fom3 was previously believed to stereospecically methylate at C2 of 2-HEP (19a) to yield S-2-hydroxypropylphosphonate (2-HPP) (26). However, enzymatic activity and mechanism of Fom3 was not completed elucidated, 67,68 because incubation of the reconstituted Fom3 with 2-HEP (19a) generated an uncharacterized product.…”