Biosynthesis of Fosfomycin, Re-Examination and Re-Confirmation of a Unique Fe(II)- and NAD(P)H-Dependent Epoxidation Reaction

Abstract

Abstract(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the epoxide ring closure of (S)-HPP to form fosfomycin, a clinically useful antibiotic. Early investigation showed that its activity can be reconstituted with Fe(II), FMN, NADH, and O 2 , and identified HppE as a new type of mononuclear non-heme iron-dependent oxygenase involving high valent iron-oxo species in the catalysis. However, a recent study showed that the Zn(II)-reconstituted HppE is active, and HppE exhibits modest affinity for FMN. Thus, a new mechanism is proposed in which the active site bound Fe 2+ or Zn 2+ serves as a Lewis acid to activate the 2-OH group of (S)-HPP, and the epoxide ring is formed by the attack of the 2-OH group at C-1 coupled with the transfer of the C-1 hydrogen as a hydride ion to the bound FMN. To distinguish between these mechanistic discrepancies, we reexamined the bioautography assay, the basis for the alternative mechanism, and showed that Zn(II) cannot replace Fe(II) in the HppE reaction, and NADH is indispensable. Moreover, we demonstrated that the proposed role for FMN as a hydride acceptor is inconsistent with the finding that FMN cannot bind to HppE in the presence of substrate. In addition, using a newly developed HPLC assay we showed that several non-flavin electron mediators could replace FMN in the HppE-catalyzed epoxidation. Taken together, these results argue against the newly proposed "nucleophilic displacement-hydride transfer" mechanism, but are fully consistent with the previously proposed iron-redox mechanism for HppE catalysis, which is unique within the mononuclear non-heme iron enzyme superfamily.Fosfomycin (1) is a clinically useful antibiotic (1) for the treatment of lower urinary tract infections (2) and limb-threatening diabetic foot infections (3). It is also effective against methicillin-resistant (4) and vancomycin-resistant (5) strains of Staphylococcus aureus. The antimicrobial activity of fosfomycin has been attributed 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 cell wall (6,7).Fosfomycin is biosynthetically derived from (S)-2-hydroxypropylphosphonic acid (2, (S)-HPP) (8,9). The conversion of (S)-HPP to fosfomycin (1) is catalyzed by HPP epoxidase (HppE) (10,11). A mononuclear non-heme iron in HppE active site is essential for enzyme activity (12). Coordination of the iron by His138, Glu142 and His180, the 2-H-1-D/E facial triad, was first implicated by sequence alignment (13) and site-directed mutagenesis studies (14), and was later confirmed by an X-ray crystal structure (15). Earlier research also showed that molecular oxygen is essential for the reaction (11,12). However, no oxygen atoms from O 2 is incorporated into the fosfomycin product (9,11,12). Instead, the oxygen atom of the epoxy *To whom correspondence should be addressed. Fax: 512-471-2746, E-mail: h.w.liu@mail.utexas NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author M...

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

“…Further analysis revealed that Zn 2ϩ strongly inhibited LipL activity (supplemental Fig. S12), and therefore co-purification with Zn 2ϩ is likely due to adventitious binding as observed with other non-heme, iron-dependent oxygenases (28). Inhibition by divalent transition metals has been reported for TauD (16) and clavaminic acid synthase (29).…”

Antibiotics in Urology – New Essentials

“…Fosfomycin (cis-(1R,2S)-epoxypropylphosphonic acid) is an oxirane antibiotic unrelated to other substances and is produced as a secondary metabolite by Streptomyces and Pseudomonas spp [14]. (S)-2-hydroxypropylphosphonic acid epoxidase catalyzes the epoxide ring closure of (S)-2-hydroxypropylphosphonic acid to form fosfomycin in an iron-redox mechanism [34]. Hydroxypropylphosphonic acid epoxidase represents a new subfamily of non-haem mononuclear iron enzymes that respond to its substrates with a conformational change that protects the radical-based intermediates formed during catalysis [35].…”

Determination of the Substrate Binding Mode to the Active Site Iron of (S)-2-Hydroxypropylphosphonic Acid Epoxidase Using 17O-Enriched Substrates and Substrate Analogues

“…The crude products were purified by flash silica gel column chromatography with hexanes/ethyl acetate (10:1 to 1:1) to give diethyl isobutylphosphonate (11) in 90% yield. A portion of 11 (0.5 g, 2.6 mmol) was then incubated with trimethylsilyl bromide (TMSBr, 0.5 mL, 3.8 mmol) and allyltrimethylsilane (0.25 mL, 1.6 mmol) for 24 h at room temperature.…”

“…The purified HppE, after reconstitution, contains a mononuclear non-heme iron that is essential and cannot be replaced by other divalent ions (10,11). The in vitro activity of HppE also requires NAD(P)H, dioxygen, and an auxiliary electron mediator to transfer electrons from NAD(P)H to the enzyme active site (8,9,11). While this mediator's role can be fulfilled by small molecule electron carriers, such as FMN, FAD, riboflavin, and benzyl viologen (11), the physiological mediator is expected to be a reductase, which has not yet been identified.…”