Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.

The biosynthesis of fosfomycin, an oxirane antibiotic in clinical use, involves a unique epoxidation catalyzed by (S)-2-hydroxypropylphosphonic acid epoxidase (HPPE). The reaction is essentially dehydrogenation of a secondary alcohol. A high-resolution crystallographic analysis reveals that the HPPE subunit displays a two-domain combination. The C-terminal or catalytic domain has the cupin fold that binds a divalent cation, whereas the N-terminal domain carries a helix-turn-helix motif with putative DNA-binding helices positioned 34 A apart. The structure of HPPE serves as a model for numerous proteins, of ill-defined function, predicted to be transcription factors but carrying a cupin domain at the C terminus. Structure-reactivity analyses reveal conformational changes near the catalytic center driven by the presence or absence of ligand, that HPPE is a Zn(2+)/Fe(2+)-dependent epoxidase, proof that flavin mononucleotide is required for catalysis, and allow us to propose a simple mechanism that is compatible with previous experimental data. The participation of the redox inert Zn(2+) in the mechanism is surprising and indicates that Lewis acid properties of the metal ions are sufficient to polarize the substrate and, aided by flavin mononucleotide reduction, facilitate the epoxidation.

The biosynthetic pathway of the clinically important antibiotic fosfomycin uses enzymes that catalyse reactions without precedent in biology. Among these is hydroxypropylphosphonic acid epoxidase, which represents a new subfamily of non-haem mononuclear iron enzymes. Here we present six X-ray structures of this enzyme: the apoenzyme at 2.0 A resolution; a native Fe(II)-bound form at 2.4 A resolution; a tris(hydroxymethyl)aminomethane-Co(II)-enzyme complex structure at 1.8 A resolution; a substrate-Co(II)-enzyme complex structure at 2.5 A resolution; and two substrate-Fe(II)-enzyme complexes at 2.1 and 2.3 A resolution. These structural data lead us to suggest how this enzyme is able to recognize and respond to its substrate with a conformational change that protects the radical-based intermediates formed during catalysis. Comparisons with other family members suggest why substrate binding is able to prime iron for dioxygen binding in the absence of alpha-ketoglutarate (a co-substrate required by many mononuclear iron enzymes), and how the unique epoxidation reaction of hydroxypropylphosphonic acid epoxidase may occur.

We present here the crystal structures of fosfomycin resistance protein (FomA) complexed with MgATP, with ATP and fosfomycin, with MgADP and fosfomycin vanadate, with MgADP and the product of the enzymatic reaction, fosfomycin monophosphate, and with ADP at 1.87, 1.58, 1.85, 1.57, and 1.85 ? resolution, respectively. Structures of these complexes that approximate different reaction steps allowed us to distinguish the catalytically active conformation of ATP and to reconstruct the model of the MgATP�Pfosfomycin complex. According to the model, the triphosphate tail of the nucleotide is aligned toward the phosphonate moiety of fosfomycin, in contest to the previously published MgAMPPNP complex, with the attacking fosfomycin oxygen positioned 4 ? from the �^-phosphorus of ATP. Site-directed mutagenesis studies and comparison of these structures with that of homologous N-acetyl-l-glutamate and isopentenyl phosphate kinases allowed us to propose a model of phosphorylation of fosfomycin by FomA enzyme. A Mg cation ligates all three phosphate groups of ATP and together with positively charged K216, K9, K18, and H58 participates in the dissipation of negative charge during phosphoryl transfer, indicating that the transferred phosphate group is highly negatively charged, which would be expected for an associative mechanism. K216 polarizes the �^-phosphoryl group of ATP. K9, K18, and H58 participate in stabilization of the transition state. D150 and D208 play organizational roles in catalysis. S148, S149, and T210 participate in fosfomycin binding, with T210 being crucial for catalysis. Hence, it appears that as in the homologous enzymes, FomA-catalyzed phosphoryl transfer takes place by an in-line predominantly associative mechanism.

Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP). Here we report the in vitro reconstitution of this unprecedented C(sp(3))-C(sp(3)) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(ii)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His-1-carboxylate mononuclear non-haem iron family of enzymes.

The fosfomycin resistance protein FomA inactivates fosfomycin by phosphorylation of the phosphonate group of the antibiotic in the presence of ATP and Mg(II). We report the crystal structure of FomA from the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis in complex with diphosphate and in ternary complex with the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP), Mg(II), and fosfomycin, at 1.53 and 2.2 angstroms resolution, respectively. The polypeptide exhibits an open alphabetaalpha sandwich fold characteristic for the amino acid kinase family of enzymes. The diphosphate complex shows significant disorder in loops surrounding the active site. As a result, the nucleotide-binding site is wide open. Binding of the substrates is followed by the partial closure of the active site and ordering of the alpha2-helix. Structural comparison with N-acetyl-L-glutamate kinase shows several similarities in the site of phosphoryl transfer: 1) preservation of architecture of the catalytical amino acids of N-acetyl-L-glutamate kinase (Lys9, Lys216, and Asp150 in FomA); 2) good superposition of the phosphate acceptor groups of the substrates, and 3) good superposition of the diphosphate molecule with the beta- and gamma-phosphates of AMPPNP, suggesting that the reaction could proceed by an associative in-line mechanism. However, differences in conformations of the triphosphate moiety of AMPPNP molecules, the long distance (5.1 angstroms) between the phosphate acceptor and donor groups in FomA, and involvement of Lys18 instead of Lys9 in binding with the gamma-phosphate may indicate a different reaction mechanism. The present work identifies the active site residues of FomA responsible for substrate binding and specificity and proposes their roles in catalysis.

Fosfomycin is a broad-spectrum antibiotic that is useful against multi-drug resistant bacteria. Although its biosynthesis was first studied over 40 years ago, characterization of the penultimate methyl transfer reaction has eluded investigators. The enzyme believed to catalyze this reaction, Fom3, has been identified as a radical S-adenosyl-L-methionine (SAM) superfamily member. Radical SAM enzymes use SAM and a four-iron, four-sulfur ([4Fe-4S]) cluster to catalyze complex chemical transformations. Fom3 also belongs to a family of radical SAM enzymes that contain a putative cobalamin-binding motif, suggesting that it uses cobalamin for methylation. Here we describe the first biochemical characterization of Fom3 from Streptomyces wedmorensis. Since recombinant Fom3 is insoluble, we developed a successful refolding and iron-sulfur cluster reconstitution procedure. Spectroscopic analyses demonstrate that Fom3 binds a [4Fe-4S] cluster which undergoes a transition between a +2 "resting" state and a +1 active state characteristic of radical SAM enzymes. Site-directed mutagenesis of the cysteine residues in the radical SAM CxxxCxxC motif indicates that each residue is essential for functional cluster formation. We also provide preliminary evidence that Fom3 adds a methyl group to 2-hydroxyethylphosphonate (2-HEP) to form 2-hydroxypropylphosphonate (2-HPP) in an apparently SAM-, sodium dithionite-, and methylcobalamin-dependent manner.

Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that uses dioxygen to catalyze the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid (S-HPP) in the biosynthesis of the antibiotic fosfomycin. Additionally, the enzyme converts the R-enantiomer of the substrate (R-HPP) to 2-oxo-propylphosphonic acid. To probe the mechanism of HppE regiospecificity, we determined three X-ray structures: R-HPP with inert cobalt-containing enzyme (Co(II)-HppE) at 2.1 ? resolution; R-HPP with active iron-containing enzyme (Fe(II)-HppE) at 3.0 ? resolution; and S-HPP-Fe(II)-HppE in complex with dioxygen mimic NO at 2.9 ? resolution. These structures, along with previously determined structures of S-HPP-HppE, identify the dioxygen binding site on iron and elegantly illustrate how HppE is able to recognize both substrate enantiomers to catalyze two completely distinct reactions.

The nucleotide sequence of the Streptomyces hygroscopicus gene encoding P-methyltransferase, catalyzing the formation of a carbon-phosphorus bond, involved in bialaphos biosynthesis, has been determined. The amino-acid sequence deduced from the nt sequence, shows homology with those of magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (Mg-ProtoMe cyclase) of Rhodobacter capsulatus and the enzyme catalyzing the methylation of the aldehyde carbon of phosphonoacetaldehyde in fosfomycin biosynthesis.

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The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.

Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, etc. Its biosynthesis starts with the formation of a carbon-phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 N-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 C-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism via such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family.