Structure and reactivity of hydroxypropylphosphonic acid epoxidase in fosfomycin biosynthesis by a cation- and flavin-dependent mechanism

Abstract

The biosynthesis of fosfomycin, an oxirane antibiotic in clinical use, involves a unique epoxidation catalyzed by (S)-2-hydroxypropylphosphonic acid epoxidase (HPPE). The reaction is essentially dehydrogenation of a secondary alcohol. A high-resolution crystallographic analysis reveals that the HPPE subunit displays a two-domain combination. The C-terminal or catalytic domain has the cupin fold that binds a divalent cation, whereas the N-terminal domain carries a helix-turn-helix motif with putative DNA-binding helices positioned 34 Å apart. The structure of HPPE serves as a model for numerous proteins, of ill-defined function, predicted to be transcription factors but carrying a cupin domain at the C terminus. Structure-reactivity analyses reveal conformational changes near the catalytic center driven by the presence or absence of ligand, that HPPE is a Zn 2؉ ͞Fe 2؉ -dependent epoxidase, proof that flavin mononucleotide is required for catalysis, and allow us to propose a simple mechanism that is compatible with previous experimental data. The participation of the redox inert Zn 2؉ in the mechanism is surprising and indicates that Lewis acid properties of the metal ions are sufficient to polarize the substrate and, aided by flavin mononucleotide reduction, facilitate the epoxidation.enzyme ͉ epoxidation ͉ iron ͉ zinc

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

“…Instead, it is believed to function as a surrogate in the iron-redox mechanism for an electron-transfer protein involved in the catalysis in vivo. Interestingly, a K d of 10 μM for FMN to HppE was determined by McLuskey et al (28), which was cited as an indication for a specific binding of FMN in HppE in support of the proposed nucleophilic displacementhydride transfer mechanism.…”

“…The HppE used by McLuskey et al is a fusion protein with a N-terminal His 6 -tag, and was purified by Ni-NTA affinity chromatography (28). In contrast, the HppE protein used in all our work does not contain an affinity tag and was purified as an apo-protein because DTT and EDTA were added to purification buffers to remove all metal ions (12).…”

“…Interestingly, the His 6 -tagged HppE purified by McLuskey et al was a mixture of 80% apoenzyme and 20% Zn(II)-containing enzyme (28). No iron was found in their protein, and no color development was noted.…”

“…It serves as an electron mediator in the iron-redox mechanism, and has been proposed to be the hydride acceptor in the nucleophilic displacement-hydride transfer mechanism (28). Since the translated sequence of HppE lacks an ADP βαβ-binding fold characteristic for flavin-dependent enzymes, and reconstitution failed to incorporate FMN in the active site, FMN has not been considered to be a true coenzyme of HppE (11).…”

“…In a recent study, McLuskey et al found that the purified recombinant HppE is active when reconstituted with Fe 2+ or Zn 2+ (28). They also showed that HppE exhibits modest affinity for FMN which is required for activity.…”