Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme

Abstract

The biosynthetic pathway of the clinically important antibiotic fosfomycin uses enzymes that catalyse reactions without precedent in biology. Among these is hydroxypropylphosphonic acid epoxidase, which represents a new subfamily of non-haem mononuclear iron enzymes. Here we present six X-ray structures of this enzyme: the apoenzyme at 2.0 Å resolution; a native Fe(ii)-bound form at 2.4 Å resolution; a tris(hydroxymethyl)aminomethane–Co(ii)-enzyme complex structure at 1.8 Å resolution; a substrate–Co(ii)-enzyme complex structure at 2.5 Å resolution; and two substrate–Fe(ii)-enzyme complexes at 2.1 and 2.3 Å resolution. These structural data lead us to suggest how this enzyme is able to recognize and respond to its substrate with a conformational change that protects the radical-based intermediates formed during catalysis. Comparisons with other family members suggest why substrate binding is able to prime iron for dioxygen binding in the absence of α-ketoglutarate (a co-substrate required by many mononuclear iron enzymes), and how the unique epoxidation reaction of hydroxypropylphosphonic acid epoxidase may occur.

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Figure 1: Fosfomycin biosynthesis.
Figure 2: Overall structure of Fe( ii )-HppE.
Figure 3: HppE active sites displayed with 2 Fo - Fc maps contoured from 1.0–1.5σ.
Figure 4: Structural insights into catalysis.
Figure 5: Schematic of interactions of bidentate-bound S -HPP with protein, water and Fe( ii).
Figure 6: Possible mechanisms for HppE.

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Acknowledgements

This research is supported in part by the National Institutes of Health (C.L.D. and H-W.L.), the National Institute of Environmental Health Sciences (L.J.H.), the Searle Scholars Program (C.L.D.), Alfred P. Sloan Foundation (C.L.D.), and a Lester Wolfe Predoctoral Fellowship (L.J.H.). Synchrotron facilities are funded by the NIH National Center of Research Resources (Advanced Photon Source NE-CAT 8BM) and the US Department of Energy, Division of Materials Sciences and Division of Chemical Sciences (National Synchrotron Light Source, Brookhaven National Laboratory).

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Correspondence to Catherine L. Drennan.

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Competing interests

Atomic coordinates for the following structures have been deposited in the Protein Data Bank: apo-HppE (1ZZ6), Fe(ii)-HppE (1ZZ9), form-1 S-HPP–Fe(ii)-HppE (1ZZ7), form-2 S-HPP–Fe(ii)-HppE (1ZZ8), Tris–Co(ii)-HppE (1ZZC) and S-HPP–Co(ii)-HppE (1ZZB). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Site-directed mutagenesis, protein purification, assays, crystallization and data collection.

Supplementary Figure S1

Stereoviews of HppE active sites displayed with 2Fo-Fc maps contoured from 1.0-1.5ω .

Supplementary Figure S2

Structural insights into catalysis (stereoviews).

Supplementary Figure S3

Stereoviews of S-HPP-Co(II)-HppE active sites displayed with 2Fo-Fc maps contoured from 1.0-1.5ω.

Supplementary Tables

Supplementary Table S1 details data and refinement statistics for Co and Apo HppE structures. Supplementary Table S2 details data and refinement statistics for Fe HppE structures

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Higgins, L., Yan, F., Liu, P. et al. Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme. Nature 437, 838–844 (2005). https://doi.org/10.1038/nature03924

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