Characterization and structure of DhpI, a phosphonate O-methyltransferase involved in dehydrophos biosynthesis

Abstract

Phosphonate natural products possess a range of biological activities as a consequence of their ability to mimic phosphate esters or tetrahedral intermediates formed in enzymatic reactions involved in carboxyl group metabolism. The dianionic form of these compounds at pH 7 poses a drawback with respect to their ability to mimic carboxylates and tetrahedral intermediates. Microorganisms producing phosphonates have evolved two solutions to overcome this hurdle: biosynthesis of monoanionic phosphinates containing two P-C bonds or esterification of the phosphonate group. The latter solution was first discovered for the antibiotic dehydrophos that contains a methyl ester of a phosphonodehydroalanine group. We report here the expression, purification, substrate scope, and structure of the O-methyltransferase from the dehydrophos biosynthetic gene cluster. The enzyme utilizes S-adenosylmethionine to methylate a variety of phosphonates including 1-hydroxyethylphosphonate, 1,2-dihydroxyethylphosphonate, and acetyl-1-aminoethylphosphonate. Kinetic analysis showed that the best substrates are tripeptides containing as C-terminal residue a phosphonate analog of alanine suggesting the enzyme acts late in the biosynthesis of dehydrophos. These conclusions are corroborated by the X-ray structure that reveals an active site that can accommodate a tripeptide substrate. Furthermore, the structural studies demonstrate a conformational change brought about by substrate or product binding. Interestingly, the enzyme has low substrate specificity and was used to methylate the clinical antibiotic fosfomycin and the antimalaria clinical candidate fosmidomycin, showing its promise for applications in bioengineering.

Fig. 1.

Examples of phosphonate natural products. (A) Chemical structures of fosfomycin, fosmidomycin, FR900098, rhizocticin A, and phosphinothricin. (B) Proposed biosynthetic pathway of dehydrophos (19). Biosynthetic steps that have been confirmed either genetically or biochemically are represented in solid arrows and proposed steps are shown in dashed arrows.

Fig. 2.

Phosphonates tested as substrates for methylation by DhpI. (A) Chemical structures of various substrate candidates. (B) Formation of (±)-Gly-Leu-AlaP-OMe analyzed by HPLC coupled to APCI/MS. (C) Michaelis–Menten curve obtained by using variable concentrations of Gly-Leu-L-AlaP (open circles) and Gly-Leu-D-AlaP (closed circles) and a fixed SAM concentration of 3 mM.

Fig. 3.

(A) Ribbon diagram for the overall structure of DhpI monomer with SAM and a sulfate anion. The core methyltransferase domain is shown in pink and the DhpI-specific insertions, consisting of the capping helix and β-hairpin insert, are highlighted in blue. The SAM cofactor is shown in green. (B) Structure of the DhpI dimer showing the domain-swapped interactions between the SAM-binding site of one monomer (shown in pink and blue) and the capping helix and β-hairpin insertion from another monomer (shown in gray and green). (C) Close-up view of the composite active site in the DhpI-SAM- structure. The sulfate anion is shown in orange, residues that contact the sulfate are shown in yellow, and the intersubunit interaction between Tyr29 and Glu155 is shown in yellow and white, respectively.

Fig. 4.

Comparison of cocrystal structures with substrates and product. (A) Ribbon diagram for the overall structure of DhpI-SAH complex. Regions of the polypeptide that undergo a conformational shift, relative to the DhpI-SAM- structure are shown in pink. (B) Close-up view of the composite active site in the DhpI-SAM- structure showing the interaction between two monomers, colored in pink and green. Active site residues from the pink monomer are shown in yellow and those from the capping helix from the green monomer are shown in green. (C) A close-up view of the equivalent region in the DhpI-SAH structure, showing the reorganization of the active site residues Tyr29 and Arg168.

Fig. 5.

Methylation of useful phosphonates. Extracted ion chromatograms showing the complete methylation of fosmidomycin and fosfomycin by DhpI. See Fig. S2 for MS spectra. (A) Fosmidomycin (RT = 5.9, m/z 184) was fully converted to fosmidomycin-OMe (RT = 9.4 min, m/z 198). (B) Fosfomycin (RT = 6.1 min, m/z 139) was fully converted to fosfomycin-OMe (RT = 9.1 min, m/z 153). N-Acetyl aspartic acid (AcAsp) was used as internal standard (IS).