Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis

Abstract

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.

Consecutive radical S -adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis

“…The relative stoichiometry of iron and sulfide bound by the as-isolated protein was determined to be 7.4 ± 1.4 and 3.7 ± 0.8 per polypeptide, respectively, consistent with the presence of a [4Fe-4S] cluster. Apparent excess iron has been observed with other RS enzymes (11,14), and was similarly seen in an alanine variant control.…”

“…1). Although other RS cobalamin-dependent enzymes that act on carbon have been investigated previously (9)(10)(11)(12), to our knowledge this is the first in vitro characterization of a RS methylase acting sequentially to form a side chain.…”

“…2A). A pantetheinyl moiety was selected for the side chain at C2 (10)(11)(12), in keeping with the OA-6129 series of metabolites that has been isolated from carbapenem producers (13).…”

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

“…A subgroup of recently characterized radical SAM methyltransferases involved in secondary metabolite biosynthesis in bacteria contain a vitamin B 12 (cobalamin)-binding motif and require cobalamin for activity (13,(31)(32)(33)(34). These enzymes are thought to use one molecule of SAM in typical radical SAM fashion to produce Ado-CH 2 · for substrate radical formation (see Fig.…”

The Extracellular Heme-binding Protein HbpS from the Soil Bacterium Streptomyces reticuli Is an Aquo-cobalamin Binder

“…In streptomycetes there are Cbl-dependent enzymes that catalyze a set of modifications to peptides or polyketides or other chemical backbones during the biosynthesis of antibiotics. For instance, the methylation of the antibiotics clorobiocin and fosfomycin is a cobalamin-dependent reaction (17,18). Looking further afield, it has been reported that Cbl interacts with a riboswitch to regulate the expression of the ribonucleotide reductase nrdABS operon in Streptomyces coelicolor A3(2) (19).…”