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

Abstract

Abstract(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) is an O 2 -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 analogs designed to probe the basis for this specificity. In past studies, NO has been used as an O 2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O 2 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 17 O (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)-analog 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 analogs 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. † This work was supported in part by the National Institutes of Health Grants (GM40541 to H. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe epoxide ring is a common structural element found in many natural products. The inherent strain energy associated with the small ring renders it reactive with enzyme nucleophiles. This has allowed many epoxide-containing compounds to be used as enzyme inhibitors and therapeutic agents. A representative example is fosfomycin ((1R, 2S)-epoxypropylphosphonic acid, 1), an antibiotic used to treat lower urinary tract infections (1,2). The biological target of fosfomycin has been identified as UDP-α-D-GlcNAc-O-enolpyruvoyl 1 transferase (3), which catalyzes the attachment of phosphoenolpyruvate to UDP-α-D-GlcNAc, a key step in the assembly of the peptidoglycan layer within the bacterial cell wall.In most cases, the oxirane moiety of epoxide-containing natural products is b...

Purification and Characterization of the Epoxidase Catalyzing the Formation of Fosfomycin from Pseudomonas syringae

“…However, the heterogeneity observed in the EPR spectrum of the ternary complex of Ps -HppE suggests that substrate binding to the active site Fe(II) ion in two different conformations. Our recent isotope labeling study showed that the uniform EPR signal observed for the ternary complex of Sw -HppE is a result of substrate coordination to the Fe(II) ion in a bidentate fashion (39). This same species is also observed with Ps -HppE, although it represents only 30% of the total iron.…”

Enzymatic Chemistry of Cyclopropane, Epoxide, and Aziridine Biosynthesis

“…207,208 EPR analysis of anaerobic, enzyme-bound Fe II -NO complexes in the presence and absence of ( S )-HPP and various substrate analogues confirmed that both the C2-OH and phosphonate functional groups of ( S )-HPP were required for bidentate binding. 204,215 The direct coordination of the negatively charged phosphonate group to the iron center in this binding mode likely helps activate Fe II towards O 2 binding and may also assist in the formation of higher iron oxidation states for substrate oxidation. 216 Overall, the crystallographic and EPR studies shed light on how HppE is able to bind HPP, prime iron for O 2 binding, and shield reactive intermediates from solvent.…”

Substrate activation by iron superoxo intermediates

“…Liu and coworkers reconstituted the activity of the HPP epoxidase (HppE) in vitro [38] and showed that the enzyme requires Fe(II), O 2 , an electron carrier (either a general reductase or catalytic amounts of FMN) and NADH as reductant, but not α-ketoglutarate. Subsequent spectroscopic, mechanistic, and structural investigations established that the enzyme is a mononuclear non-heme-iron-dependent oxidase [22,39–41]. …”

“…The latter binding mode is associated with a more closed conformation that appears to shield the active site from solvent and may help protect reactive intermediates. Further support for productive bidentate HPP binding has been provided by EPR studies with 17 O-labeled substrate and using NO as mimic of oxygen [41]. The bidentate binding of HPP resembles the binding geometry of α-ketoglutarate (α-KG) in other members of the cupin family [42,43], and analogously the bidentate binding by HPP may serve to facilitate the normally unfavorable reaction of ferrous iron with molecular oxygen [22].…”