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May 24, 2006

Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme

L.J. Higgins^1,*, F. Yan², P. Liu², H.-w. Liu², C.L. Drennan^1
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA; ²Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX; ^*Currently a Harvard Medical School Fellow, Cambridge, MA

The mononuclear iron enzyme S-(2)-hydroxypropylphosphonic acid epoxidase (HppE), from Streptomyces wedmorensis, uses O₂ to catalyze the formation of the broad-spectrum antibiotic fosfomycin (which inhibits bacterial cell-wall peptidoglycan biosynthesis) from S-(2)-hydroxypropylphosphonic acid (S-HPP). The reaction is a two-electron oxidation and is mechanistically atypical because it is independent of any cofactor or co-substrate and results in the incorporation of the hydroxyl oxygen of the substrate, rather than an atom of O₂, into the epoxide ring. The x-ray crystal structures of six forms of HppE - apo-HppE, Fe(II)-HppE, tris(hydroxymethyl)aminomethane-Co(II)-HppE complex, S-HPP-Co(II)-HppE complex, and two S-HPP-Fe(II)-HppE complexes - were solved using data collected in part at the NSLS. The purpose of this was to gain insight into the mechanism of this unique enzyme.

Catherine Drennan

Luke Higgins

Mononuclear non-heme iron enzymes use their metal cofactor to activate dioxygen (O₂) for difficult redox processes. One of these enzymes, S-(2)-Hydroxypropylphosphonic acid epoxidase (HppE), from Streptomyces wedmorensis (Figure 1, overall structure), employs its mononuclear iron center and molecular oxygen for the two-electron oxidation of S-(2)-hydroxypropylphosphonic acid (S-HPP) to catalyze the formation of the antibiotic (1R,2S)-(1,2-epoxypropyl)phosphonic acid (fosfomycin). This reaction is essentially a dehydrogenation reaction (loss of hydrogen). In order to balance the four-electron reduction of oxygen to water, we have proposed a putative two-electron reductant (or reductase). Fosfomycin is an unusual C-P-bond-containing epoxide that covalently modifies UDP-GlcNAc enolpyruvyl transferase, consequently inhibiting bacterial cell-wall peptidoglycan biosynthesis and bacterial growth. Since it accumulates in the kidneys and bladder, fosfomycin has been used clinically for the treatment of lower-urinary-tract infections. The structures of the apo-HppE (metal-free), native Fe(II)-HppE, tris(hydroxymethyl)aminomethane (Tris)-Co(II)-HppE complex, S-HPP-Co(II)-HppE complex, and two S-HPP-Fe(II)-HppE complexes (form 1 and form 2) were solved in order to better understand the epoxidation mechanism of this enzyme. It is interesting to note that the Tris molecules in the Tris-Co(II)-HppE structure result from the Tris buffer used in the crystallization solution.

Figure 1. Tetrameric structure of HppE.

Selenomethionine (SeMet) derivatization was used to obtain phase information for these protein structures. Initial experimental phases were determined from an x-ray dataset of Tris-Co(II)-SeMet-HppE collected at the wavelength for the Se absorption peak (0.9791 Å) on NSLS beamline X26C. This dataset was refined to 2.5 Å in space group P6₅22 and the resulting model was further refined against a 1.8 Å native Tris-Co(II)-HppE dataset that was obtained at 0.9791 Å on Advanced Photon Source beamline 8BM. All subsequent structures were determined from these initial models.

Figure 2. A portion of the structure called the cantilever (red) closes over the active site iron (brown) and substrate (ball-and-stick).

Scheme 1. Possible reaction mechanisms for HppE.

The structures of S-HPP-Fe(II)-HppE complexes (form 1 and form 2) confirm the direct binding of the substrate, S-HPP, to the iron and show the existence of two binding modes, a monodentate mode and a bidentate mode. These two modes are explained by a two-step binding process: (i) S-HPP first binds in a monodentate fashion via the oxygen atom of the phosphonic acid group, resulting in displacement of a water molecule; (ii) The subsequent rotation of the substrate allows for bidentate coordination of S-HPP to Fe(II). A -hairpin-like structure, formed by -strands 2 and 3, acts as a cantilever that responds to the bidentate positioning of the substrate and adopts a closed catalytic conformation to cover the hydrophobic portion of the substrate bound in the active site (Figure 2). The negative charge on the substrate is expected to enhance the reactivity of the diiron center toward oxygen, a role that co-substrates play in other mononuclear iron proteins. The addition of oxygen to the only open coordination site on Fe(II) of the bidentate-S-HPP complex appears to occur through a very small channel created at the interface of the - and - domains. Once O₂ is bound, abstraction of the C1 hydrogen atom could occur by a Fe(IV)-oxo intermediate (Scheme 1, pathway A) or by a Fe(III)-hydroperoxide intermediate (Scheme 1, pathway B). This results in the formation of a transient-substrate radical intermediate that undergoes cyclization to yield fosfomycin.

BEAMLINE
X26C

FUNDING
National Institutes of Health
National Institute of Environmental Health Sciences
Searle Scholars Program
Alfred P. Sloan Foundation
A Lester Wolfe Predoctoral Fellowship

PUBLICATION
L.J. Higgins, F. Yan, P. Liu, H.-W. Liu, and C. L. Drennan, Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme, Nature, 437, 838-844 (2005).

FOR MORE INFORMATION
Catherine L. Drennan
Department of Chemistry
Massachusetts Institute of Technology
Cambridge, MA
Email: cdrennan@mit.edu