Evidence for radical-mediated catalysis by HppE: a study using cyclopropyl and methylenecyclopropyl substrate analogues

Abstract

(S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that catalyzes the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid ((S)-HPP) in the biosynthesis of the antibiotic fosfomycin. HppE also recognizes (R)-2-hydroxypropylphosphonic acid ((R)-HPP) as a substrate and converts it to 2-oxo-propylphosphonic acid. To probe the mechanisms of these HppE-catalyzed oxidations, cyclopropyl- and methylenecyclopropyl-containing compounds were synthesized and studied as radical clock substrate analogues. Enzymatic assays indicated that the (S)- and (R)-isomers of the cyclopropyl-containing analogues were efficiently converted to epoxide and ketone products by HppE, respectively. In contrast, the ultrafast methylenecyclopropyl-containing probe inactivated HppE, consistent with a rapid radical-triggered ring-opening process that leads to enzyme inactivation. Taken together, these findings provide, for the first time, experimental evidence for the involvement of a C2-centered radical intermediate with a lifetime on the order of nanoseconds in the HppE-catalyzed oxidation of (R)-HPP.

Figure 1

¹H-NMR end-point assay of HppE with (R)- and (S)-7. (A) Initial time point recorded 3 min after incubation of substrate with enzyme. (B) 36 min time point showing >90% conversion of (S)-7 to 13. (C) 36 min time point showing >90% conversion of (R)-7 to 15. (See Figure S4-1, S4-2 for details).

Figure 2

¹H-NMR assay of HppE with 16. Spectra were recorded at different time points. Only the region (5.1–5.4 ppm) of the olefinic protons is shown. No further consumption of 16 was observed after ~10 min.

Scheme 1

Proposed mechanism for the oxidation of (S)- and (R)-HPP by HppE. (A) Conversion of (S)-HPP (2) to fosfomycin (1). (B) Conversion of (R)-HPP (3) to 2-oxopropylphosphonic acid (4).

Scheme 2

Synthetic schemes for the preparation of (S)- and (R)-7.

Scheme 3

Conversion of (S)-7 to 13 and (R)-7 to 15 by HppE.

Scheme 4

Synthetic scheme for the preparation of 16.

Scheme 5

Proposed reactions of (2S)- and (2R)-16 with HppE.

Scheme 6

Proposed mechanisms for the inactivation of HppE by (2R)-16.