Mechanistic studies of an unprecedented enzyme-catalysed 1,2-phosphono-migration reaction

Abstract

(S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-haem-iron-dependent enzyme responsible for the final step in the biosynthesis of the clinically useful antibiotic fosfomycin. Enzymes of this class typically catalyse oxygenation reactions that proceed via the formation of substrate radical intermediates. By contrast, HppE catalyses an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin. Here we show that HppE also catalyses a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation probably involves an intermediary carbocation, based on observations with additional substrate analogues, such as (1R)-1-hydroxyl-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyse distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-haem-iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of new phosphonate derivatives.

Figure 1. Fosfomycin biosynthetic pathway and HppE-catalyzed conversion of various substrate analogues

(A) Formation of fosfomycin (1) from PEP (2). (B) Conversion of (R)-2-HPP (5) to the corresponding ketone 6. (C) Conversion of (S)-1-HPP to acyl phosphonate (8). (D) Conversion of (R)-1-HPP (7) to the aldehyde product (9).

Figure 2. ¹H NMR time-course for the HppE-catalyzed conversion of (A) (S)-7 to 8, and (B) (R)-7 to 9

The peak at δ 2.49 is from DMSO-d₆, and those (in black) centered between δ 2.50 and 2.65 are from NADH. The NMR signals and the contributing proton(s) are color-coded.

Figure 3. Structures of (S)-7 and (R)-7 bound to the iron center of HppE in a bidentate mode

(A) (S)-7 (carbons in blue) in F_o-F_c omit map density contoured at 6 σ (left) and its chemical structure (right), with hydrogen atoms accessible for abstraction in red and inaccessible in blue. The putative dioxygen binding site on Fe is partially occupied by water molecules (red spheres) in both of these structures (Fe-H₂O distances are 3.7 and 3.0 Å). Colors: Fe in rust, P in orange, O in red, N in blue, protein C in gray. (B) (R)-7 (carbons in green) in F_o-F_c omit map density contoured at 6 σ (left) and its chemical structure (right). Hydrogen atoms labeled as in (A).

Figure 4

(A) Stereochemistry of hydrogen atom abstraction from (R)-1-HPP (7) determined using the stereospecifically deuterated compounds 11 and 12. (B) Hypothetical mechanisms for HppE-catalyzed 1,2-phosphono migration involving cation (route a) and radical (route b) mediated rearrangements. Intermediate 14 could be generated in a manner analogous to the formation of 31 from (S)-2-HPP (see Fig. 5). (C) Model reactions to probe the involvement of radical or cation intermediates in the 1,2-phosphono migration catalyzed by HppE. The migration product is only observed when cation 19 is formed from 17 using silver-triflate. (D) HppE-catalyzed conversion of (RR)- and (RS)-23 to the imine hydrolysis product 26, consistent with the oxidation of (R)-7 to a cationic intermediate by a reactive iron-oxygen species. Inset: Compound 27, which lacks the C1 hydroxyl group, is not a substrate of HppE, indicating that the amino group is not capable of supporting the bidentate substrate coordination required for catalysis.

Figure 5

Revised mechanisms for the HppE-catalyzed epoxidation of (S)-2-HPP (4) involving C1 cation formation (route a) or O-atom rebound (route b).