Purification and Characterization of the Epoxidase Catalyzing the Formation of Fosfomycin from Pseudomonas syringae

Abstract

The final step in the biosynthesis of fosfomycin in Streptomyces wedmorensis is catalyzed by (S)-2-hydroxypropylphosphonic acid (HPP) epoxidase (Sw-HppE). A homologous enzyme from Pseudomonas syringae has recently been isolated whose encoding gene (orf3) shares relatively low sequence homology to the corresponding Sw-HppE gene. This purified P. syringae protein was determined to catalyze the epoxidation of (S)-HPP to fosfomycin and the oxidation of (R)-HPP to 2-oxopropylphosphonic acid under the same conditions as Sw-HppE. Therefore, this protein is indeed a true HPP epoxidase and is termed Ps-HppE. Like Sw-HppE, Ps-HppE was determined to be posttranslationally modified by the hydroxylation of a putative active site tyrosine (Tyr95). Analysis of the Fe(II)-center by EPR spectroscopy using NO as a spin probe and molecular oxygen surrogate reveals that Ps-HppE's metal center is similar, but not identical, to that of Sw-HppE. The identity of the rate determining step for the (S)-HPP and (R)-HPP reactions was determined by measuring primary deuterium kinetic effects, and the outcome of these results were correlated with density functional theory calculations. Interestingly, the reaction using the non-physiological substrate (R)-HPP was 1.9 times faster than that with (S)-HPP for both Ps-HppE and Sw-HppE. This is likely due to the difference in bond dissociation energy of the abstracted hydrogen atom for each respective reaction. Thus, despite low amino acid sequence identity, Ps-HppE is a close mimic of Sw-HppE, representing a second example of a non-heme iron-dependent enzyme capable of catalyzing dehydrogenation of a secondary alcohol to form a new C-O bond.Fosfomycin (1) is a clinically useful antibiotic (1) for the treatment of limb-threatening diabetic foot infections (2) and lower urinary tract infections. It has been shown to be effective against ciprofloxacin-resistant Escherichia coli (3), as well as methicillin-resistant (4) and vancomycin-resistant (5) strains of Staphylococcus aureus. The antimicrobial activity of fosfomycin is due to the inactivation of UDP-GlcNAc-3-O-enolpyruvyltransferase (MurA), which catalyzes the first committed step in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall (6,7). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptFosfomycin belongs to a steadily growing family of natural products containing a C-P bond (8) (9). Members of this family, which include fosmydomicin (10) and bialaphos (11) are all derived from phosphoenolpyruvate (PEP, 2). The C-P bonds in these compounds are formed through an intramolecular rearrangement reaction catalyzed by PEP mutase, resulting in the conversion of PEP (2) to phosphopyruvate (PnPy, 3) ( Fig. 1) (12)(13)(14). Since the equilibrium between PEP and PnPy favors PEP, the decarboxylation catalyzed by the second enzyme in the biosynthetic pathway, PnPy decarboxylase, provides the driving force to shift the equilibrium toward C-P bond formation (15). The next two steps in the pathway...

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

“…2 and Table 2). As expected from previous studies on the epoxidase responsible for the last step of fosfomycin biosynthesis in both streptomycetes (37) and pseudomonads (36,40) (Fig. 1), a gene encoding the epoxidase was present (here designated psf4).…”

“…fradiae and P. syringae. As demonstrated by the activity of the purified enzymes Psf1 and Fom4 in this and previous studies (36,40), the pathways have the first and last steps in common. Sequence alignments show that Fom1 and Fom4 from S. fradiae have 32% and 35% identity to Psf1 and Psf4, respectively (see Fig.…”

“…3). Aside from the previously demonstrated epoxidase activity of Psf4 (36,40), which converts 2-HPP to fosfomycin in the last step of the pathway, the sequences of the proteins encoded by the additional ORFs in the gene cluster do not provide immediate clues for the manner in which Pmm is converted to 2-HPP (Table 2). Psf3 shows sequence homology with an NAD-dependent 6-phosphogluconate dehydrogenase, whereas Psf5 has sequence homology with nonheme iron-dependent proteins ( Table 2).…”

“…After the decarboxylation/elimination reactions, compound 2 would tautomerize to compound 3, and this ␤-keto acid could undergo another decarboxylation to produce 2-oxopropionyl phosphonate (2-OPP) promoted by one of the ORFs of unknown function. Subsequent reduction, possibly by Psf3, would generate 2-HPP, which is the substrate for Psf4-catalyzed formation of fosfomycin (36,40). As discussed above, some of the putative intermediates in Fig.…”

Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis

“…2-HPP was synthesized by modifying a published procedure [24]. To 1 g dimethyl (5.1 mmol) 2-oxopropylphosphonate (95%, Acros Organics) was added 30 mL methanol and 1.92 g (51 mmol) sodium borohydride.…”