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Determination of the Substrate Binding Mode to the Active Site Iron of (S)-2-Hydroxypropylphosphonic Acid Epoxidase Using ¹⁷O-Enriched Substrates and Substrate Analogues^†

Feng Yan,^§ Sung-Ju Moon,^§ Pinghua Liu,^§^‡ Zongbao Zhao,^§^# John D. Lipscomb,* Aimin Liu,*^£ and Hung-wen Liu*^§

Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, Department of Biochemistry, Molecular Biology, and Biophysics and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455, and Department of Biochemistry, University of Mississippi Medical Center, University of Mississippi, 2500 N. State Street, Jackson, Mississippi 39216

Biochemistry, 2007, 46 (44), pp 12628–12638

DOI: 10.1021/bi701370e

Publication Date (Web): October 10, 2007

†

This work was supported in part by the National Institutes of Health Grants (GM40541 to H.-w.L. and GM24689 to J.D.L.) and the ORAU Faculty Enhancement Award for Life Sciences (to A.L.).

University of Texas.

Current address: Immunomedics, Inc., 300 American Rd., Morris Plains, NJ 07950.

‡

Current address: Department of Chemistry, Boston University, Boston, MA 02215.

Current address: Dalian Institute of Chemical Physics, CAS, Dalian 116023, P. R. China.

To whom correspondence should be addressed. Phone: 512-232-7811. Fax: 512-471-2746. E-mail: h.w.liu@mail.utexas.edu.

University of Minnesota.

University of Mississippi.

Abstract

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an O₂-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio- and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogues designed to probe the basis for this specificity. In past studies, NO has been used as an O₂ surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O₂ binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO−HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with ¹⁷O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analogue compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogues lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)−substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.