Structure and mechanism of enzymes involved in biosynthesis and breakdown of the phosphonates fosfomycin, dehydrophos, and phosphinothricin

Abstract

Recent years have seen a rapid increase in the mechanistic and structural information on enzymes that are involved in the biosynthesis and breakdown of naturally occurring phosphonates. This review focuses on these recent developments with an emphasis on those enzymes that have been characterized crystallographically in the past five years, including proteins involved in the biosynthesis of phosphinothricin, fosfomycin, and dehydrophos and proteins involved in resistance mechanisms.

Figure 1

Phosphorus-carbon bond formation by PEP mutase and structures of the phosphonate natural products for which the gene clusters have been determined.

Figure 2

(A) Reactions catalyzed by HEPD and HppE. (B) Structure of the HEPD-HEP complex with the active metal-containing domain colored in cyan and the vestigial domain colored in pink. The bound Cd(II) is shown as a green sphere, with protein ligands and the substrate molecule shown as stick figures. (C) Structure of the HppE-fosfomycin complex with the cupin domain colored in cyan and the novel alpha domain colored in blue. The requisite Fe(II) is colored in orange with protein ligands and the substrate molecule shown as stick figures. (D) Structure of the HEPD dimer suggesting the relevance of the vestigial repeat in forming the composite active site. (E) Structure of the HppE tetramer shown in the same orientation as that for the HEPD dimer.

Figure 3

(A) Two proposed mechanisms for the HEPD reaction. (B) The substrate analog 1-HEP is converted to acetylphosphate by HEPD. (C) Two proposed mechanisms for the HppE reaction. The phosphonate groups are shown as dianionic but could be monoprotonated. L = undefined ligand, probably water or hydroxide.

Figure 4

(A) Reaction catalyzed by DhpI and structures of several of the phosphonates that are also substrates for the enzyme. (B) Structure of the DhpI-SAM-sulfate complex showing the nucleotide binding Rossman fold in brown and the unusual insertions necessary for substrate binding in blue. The SAM co-factor and sulfate anion are shown as stick figures. (C) Structure of the DhpI-SAH complex with the nucleotide-binding domain colored in cyan and part of the novel insertion as well as a newly formed helix colored in pink. The helix of the insertion is disordered in the SAH complex.

Figure 5

A close-up view of the actives sites of (A) HppE in complex with 2-HPP, (B) HEPD in complex with 2-HEP, and (C) DhpI in complex with 2-HEP. Despite the lack of any notable sequence similarities, both HEPD and HppE use a near identical constellation of active site residues to carry out their respective reactions. Notably, both polypeptides contain composite active sites with a catalytically essential lysine residue (colored in cyan) from a different subunit interacting with the active site. A comparison of HppE, HEPD, and DhpI co-crystal structures illustrates the chemical features that are used by functionally and structurally distinct enzymes to harbor phosphonate substrates.

Figure 6

(A) Reaction catalyzed by the fosfomycin thiol transferase FosA. (B) Reaction catalyzed by the homologous FosX metalloenzyme that hydrates the antibiotic. (C) Structure of the fosfomycin inactivating enzyme FosA. (D) Close-up view of the active site of FosA. (E) Structure of the fosfomycin hydrolase FosX. (F) Close-up view of the active site of FosX revealing a glutamate residue (Glu44) that is proximal to the substrate and that is essential for catalysis. This residue is absent in FosA.