Site-directed mutagenesis and spectroscopic studies of the iron-binding site of (S)-2-hydroxypropylphosphonic acid epoxidase

Feng Yan, Tingfeng Li, John D. Lipscomb, Aimin Liu, Hung Wen Liu

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6 Scopus citations


(S)-2-Hydroxylpropanylphosphonic acid epoxidase (HppE) is a novel type of mononuclear non-heme iron-dependent enzyme that catalyzes the O2 coupled, oxidative epoxide ring closure of HPP to form fosfomycin, which is a clinically useful antibiotic. Sequence alignment of the only two known HppE sequences led to the speculation that the conserved residues His138, Glu142, and His180 are the metal binding ligands of the Streptomyces wedmorensis enzyme. Substitution of these residues with alanine resulted in significant reduction of metal binding affinity, as indicated by EPR analysis of the enzyme-Fe(II)-substrate-nitrosyl complex and the spectral properties of the Cu(II)-reconstituted mutant proteins. The catalytic activities for both epoxidation and self-hydroxylation were also either eliminated or diminished in proportion to the iron content in these mutants. The complete loss of enzymatic activity for the E142A and H180A mutants in vivo and in vitro is consistent with the postulated roles of the altered residues in metal binding. The H138A mutant is also inactive in vivo, but in vitro it retains 27% of the active site iron and nearly 20% of the wild-type activity. Thus, it cannot be unequivocally stated whether H138 is an iron ligand or simply facilitates iron binding due to proximity. The results reported herein provide initial evidence implicating an unusual histidine/carboxylate iron ligation in HppE. By analogy with other well-characterized enzymes from the 2-His-1-carboxylate family, this type of iron core is consistent with a mechanism in which both oxygen and HPP bind to the iron as a first step in the in the conversion of HPP to fosfomycin.

Original languageEnglish (US)
Pages (from-to)82-91
Number of pages10
JournalArchives of Biochemistry and Biophysics
Issue number1
StatePublished - Oct 1 2005

Bibliographical note

Funding Information:
This work was supported in part by the National Institutes of Health Grants (GM40541 to H. w.L. and GM24689 to J.D.L.). A.L. is supported by the faculty start-up of UMMC and the ORAU Faculty Enhancement Award.

Copyright 2008 Elsevier B.V., All rights reserved.


  • 2-H-1-D/E motif
  • EPR
  • Fosfomycin biosynthesis
  • Non-heme iron


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