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Structural basis of regiospecificity of a mononuclear iron enzyme in antibiotic fosfomycin biosynthesis.

Yun D, Dey M, Higgins LJ, Yan F, Liu HW, Drennan CL - J. Am. Chem. Soc. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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 regiospecificity, 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.

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The structure of S-HPP–Fe(II)–HppE in complex with NO. (a) The active site of S-HPP–Fe(II)–HppE is shown with the Fe(II) atom (rust) as a sphere and substrate, residues, and NO as sticks. An Fo – Fc omit map (green mesh) is contoured at 7.0 σ. (b) When water is refined in a position similar to that of the nitrogen of NO in the active site, shown in Figure 4a, positive Fo – Fc difference density (green mesh) at 4.0 σ indicates that water is not large enough to account for the density.
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fig4: The structure of S-HPP–Fe(II)–HppE in complex with NO. (a) The active site of S-HPP–Fe(II)–HppE is shown with the Fe(II) atom (rust) as a sphere and substrate, residues, and NO as sticks. An Fo – Fc omit map (green mesh) is contoured at 7.0 σ. (b) When water is refined in a position similar to that of the nitrogen of NO in the active site, shown in Figure 4a, positive Fo – Fc difference density (green mesh) at 4.0 σ indicates that water is not large enough to account for the density.

Mentions: With the hairpin closed, there is space available for oxygen to enter the active site in a cavity created at the interface of the helical and β-barrel domains.(15) This cavity leads to the only open coordination site in any of the substrate-bound structures, a position trans to His180. To obtain experimental evidence that this site is in fact the oxygen binding site, NO was used as an O2 mimic. Similar to previously observed structures of S-HPP–Fe(II)–HppE,(15) the structure of S-HPP–Fe(II)–HppE–NO at 2.85 Å resolution reveals bidentate coordination of the substrate to the Fe(II) center via the C2 hydroxyl and the phosphonate oxygen in all three molecules in the asymmetric unit. In one of the three monomers, there is density only for a water molecule and not for NO (Supporting Information Figure 1h). However, in the other two monomers (monomers A,B (Fe–S-HPP–NO)), NO is coordinated to the Fe(II) center as seen by omit and positive difference electron density that is consistent with a diatomic molecule (Figure 4a,b; also see Supporting Information Figure 1f,g). When a water molecule is refined into the electron density of monomers A and B instead of NO, positive difference electron density results, indicating the presence of a second atom (Figure 4b). When NO is refined in this position, no positive or negative difference electron density appears, indicating that a diatomic molecule is the correct size.


Structural basis of regiospecificity of a mononuclear iron enzyme in antibiotic fosfomycin biosynthesis.

Yun D, Dey M, Higgins LJ, Yan F, Liu HW, Drennan CL - J. Am. Chem. Soc. (2011)

The structure of S-HPP–Fe(II)–HppE in complex with NO. (a) The active site of S-HPP–Fe(II)–HppE is shown with the Fe(II) atom (rust) as a sphere and substrate, residues, and NO as sticks. An Fo – Fc omit map (green mesh) is contoured at 7.0 σ. (b) When water is refined in a position similar to that of the nitrogen of NO in the active site, shown in Figure 4a, positive Fo – Fc difference density (green mesh) at 4.0 σ indicates that water is not large enough to account for the density.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3140168&req=5

fig4: The structure of S-HPP–Fe(II)–HppE in complex with NO. (a) The active site of S-HPP–Fe(II)–HppE is shown with the Fe(II) atom (rust) as a sphere and substrate, residues, and NO as sticks. An Fo – Fc omit map (green mesh) is contoured at 7.0 σ. (b) When water is refined in a position similar to that of the nitrogen of NO in the active site, shown in Figure 4a, positive Fo – Fc difference density (green mesh) at 4.0 σ indicates that water is not large enough to account for the density.
Mentions: With the hairpin closed, there is space available for oxygen to enter the active site in a cavity created at the interface of the helical and β-barrel domains.(15) This cavity leads to the only open coordination site in any of the substrate-bound structures, a position trans to His180. To obtain experimental evidence that this site is in fact the oxygen binding site, NO was used as an O2 mimic. Similar to previously observed structures of S-HPP–Fe(II)–HppE,(15) the structure of S-HPP–Fe(II)–HppE–NO at 2.85 Å resolution reveals bidentate coordination of the substrate to the Fe(II) center via the C2 hydroxyl and the phosphonate oxygen in all three molecules in the asymmetric unit. In one of the three monomers, there is density only for a water molecule and not for NO (Supporting Information Figure 1h). However, in the other two monomers (monomers A,B (Fe–S-HPP–NO)), NO is coordinated to the Fe(II) center as seen by omit and positive difference electron density that is consistent with a diatomic molecule (Figure 4a,b; also see Supporting Information Figure 1f,g). When a water molecule is refined into the electron density of monomers A and B instead of NO, positive difference electron density results, indicating the presence of a second atom (Figure 4b). When NO is refined in this position, no positive or negative difference electron density appears, indicating that a diatomic molecule is the correct size.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

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 regiospecificity, 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.

Show MeSH
Related in: MedlinePlus