ARTICLE pubs.acs.org/JACS Structural Basis of Regiospecificity of a Mononuclear Iron Enzyme in Antibiotic Fosfomycin Biosynthesis Danny Yun, ‡ Mishtu Dey,†,‡ Luke J. Higgins,‡,^ Feng Yan,|| Hung-wen Liu,|| and Catherine L. Drennan*,†,‡,§ †Howard Hughes Medical Institute, ‡Department of Chemistry, and §Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States D)ivision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, United States bS Supporting Information ABSTRACT: Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that uses dioxygen to catalyze the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid (S-HPP) in the biosynthesis of the antibiotic fosfomycin. Additionally, the enzyme converts the R-enantiomer of the substrate (R- HPP) to 2-oxo-propylphosphonic acid. To probe the mechanism of HppE regiospe- ciﬁcity, we determined three X-ray structures: R-HPP with inert cobalt-containing enzyme (Co(II) HppE) at 2.1 Å resolution; R-HPP with active iron-containing enzyme (Fe(II) HppE) at 3.0 Å resolution; and S-HPP Fe(II) HppE in complex with dioxygen mimic NO at 2.9 Å resolution. These structures, along with previously determined structures of S-HPP HppE, identify the dioxygen binding site on iron and elegantly illustrate how HppE is able to recognize both substrate enantiomers to catalyze two completely distinct reactions. ’ INTRODUCTION Fosfomycin, or (1R,2S)-1,2-epoxypropyl-phosphonic acid, is an antibiotic used in the treatment of lower urinary tract infections1 and is eﬀective against methicillin-resistant2 and vancomycin-resistant3 strains of Staphylococcus aureus. It targets the ﬁrst committed step in cell wall biosynthesis by inhibiting the enzyme UDP-GlcNAc-3-O-enolpyruvyltransferase (MurA).4,5 Key to the reactivity of fosfomycin is the epoxide ring in its scaﬀold. While epoxide rings are found in many natural products, the way in which the fosfomycin epoxide ring is biosynthesized by microbes such as Streptomyces wedmorensis and Pseudomonas syringae may be unique.6,7 Unlike other characterized epoxidases that form epoxides through oxygen atom insertion, hydroxypro- pylphosphonic acid epoxidase (HppE) forms fosfomycin by catalyzing an oxidative cyclization reaction with retention of the substrate hydroxyl oxygen atom8 10 (Scheme 1a). Despite one report11 that HppE has some catalytic activity with zinc, recent studies show that under deﬁned conditions, Zn HppE does not produce fosfomycin, reaﬃrming this enzyme as a member of the mononuclear nonheme iron superfamily.12 HppE shares several features with other members of this superfamily, including the use of the so-called 2-His-1-carbox- ylate facial triad [His2(Glu/Asp)] to coordinate an iron atom within a conserved β-barrel fold also known as a cupin fold.9,10,13 15 With highest structural similarity to the mononuclear iron enzyme hydroxyethylphosphonate dioxygenase,16 HppE is an unusual tetramer with four β-barrels intertwined with four R-helical domains, creating four active sites at each of the R β interfaces (Figure 1).15 The substrate (S)-2-hydroxypropylpho- sphonic acid (S-HPP) binds directly to the iron center in these active sites, triggering conformational changes in the β-hairpin, or cantilever hairpin, which is directly above each facial triad.15 While other mononuclear iron enzymes can undergo confor- mational changes when substrates bind,17 19 the use of these β-strands is unique to HppE. This conformational change buries the hydrophobic portion of the substrate, reducing solvent accessibility of the active site and thus protecting high-energy iron oxygen intermediates, such as a superoxide intermediate (Fe O2 ),20 which may form during catalysis. For complete turnover, HppE also requires two exogenous electrons, and unlike many members of this superfamily, R-ketoglutarate does not serve this function. Instead, these electrons are derived from NAD(P)H and shuttled to the active site by a reductase in vivo.6,9 Another fascinating feature of HppE is that it recognizes both S-HPP and its enantiomeric substrate R-HPP and is an eﬃcient catalyst with respect to both reactions.21,22 Interestingly, R-HPP is not converted to an epoxide. Rather, this stereoisomer exclusively yields a ketone product, 2-oxopropylphosphonic acid (Scheme 1b).21,22 To probe the mechanism of regiospeciﬁcity of HppE catalysis, both enantiomers of the substrate were synthe- sized incorporating ﬂuorines in place of the C1 methylene hydrogens (S-FHPP and R-FHPP).22 S-FHPP is a strong com- petitive inhibitor of HppE activity, while R-FHPP is a substrate and is converted to 1-diﬂuoro-2-oxopropylphosphonic acid (Scheme 1c,d).22 This result suggests that the reaction path is determined by the regiospeciﬁcity of hydrogen atom abstraction; a C1 hydrogen atom is abstracted from the S-HPP substrate, and a C2 hydrogen atom is abstracted from R-HPP. A radical Received: March 21, 2011 Published: June 17, 2011 r 2011 American Chemical Society 11262 dx.doi.org/10.1021/ja2025728 | J. Am. Chem. Soc. 2011, 133, 11262–11269