New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin

Woodyer, Ryan D.; Li, Gongyong; Zhao, Huimin; Donk, Wilfred A. van der

doi:10.1039/b614678c

Cited by 114 publications

(119 citation statements)

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“…Discussion PEP mutase (PEPM) installs the P-C bond in all phosphonates for which the biosynthetic gene clusters are currently known. As part of a multidisciplinary program focused on naturally occurring phosphonates we have recently demonstrated that the gene clusters of these compounds can be readily identified using the pepM gene as a marker (3)(4)(5)(6)(7)(8)(9)(10). In addition, these studies have shown that phosphonate biosynthesis is widespread and that new clusters that encode for as yet structurally unidentified phosphonates are readily found (1).…”

Section: Resultsmentioning

confidence: 95%

“…Other members mimic carboxyl groups or the tetrahedral intermediates formed during enzymatic metabolism of these moieties. Recent studies have started to reveal nature's biosynthetic strategies toward several of these compounds, including the clinically used antibiotic fosfomycin (2)(3)(4), the antimalaria clinical candidate FR900098 (5,6), the antifungal tripeptide rhizocticin (7), and the widely used herbicide phosphinothricin (8)(9)(10)(11)(12) (Fig. 1A).…”

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confidence: 99%

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

Lee

Bae

Kuemin

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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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.antibiotics | bioengineering | conformational change | X-ray crystallography | domain-swap

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Section: Resultsmentioning

confidence: 95%

mentioning

confidence: 99%

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

Lee

Bae

Kuemin

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous studies in several laboratories have supported the fosfomycin biosynthetic pathway shown in Fig. 1 in Streptomyces fradiae and Streptomyces wedmorensis (37,50,59,60). Like in nearly all phosphonate biosynthetic pathways (39), the first committed step in fosfomycin biosynthesis in these streptomycetes is the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) (22,23,33).…”

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confidence: 99%

“…This thermodynamically unfavorable transformation is made feasible by linking it to the irreversible decarboxylation of PnPy to phosphonoacetaldehyde (PnAA) by a thiamine-dependent decarboxylase (41). Subsequent reduction to 2-hydroxyethylphosphonate (2-HEP) (51,59), methylation to generate 2-hydroxypropylphosphonate (2-HPP) (20,34,59,60), and epoxide formation (19,25,37,49,62) complete the biosynthesis of fosfomycin ( Fig. 1).…”

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Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

Kim

Metcalf

et al. 2012

Antimicrob Agents Chemother

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cFosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

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Cobalt Enzymes/Models

Kräutler

2010

Encyclopedia of Catalysis

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Vitamin B 12 and its coenzyme forms are prominent cobalt‐containing cofactors and part of the larger class of the natural tetrapyrrolic metal complexes. Such cobalt corrinoids are an important component of human nutrition, and many (other) forms of life also depend upon them. Cobalt‐containing corrinoid cofactors play basic roles in carbon fixation and energy metabolism in anaerobes, as well as in the carbon metabolism in a broad range of lower organisms. Microorganisms developed unique B 12 biosynthetic means and, indeed, are the only natural sources of the B 12 derivatives. Other organisms have evolved intricate strategies for the uptake and transport of the corrinoids. The cofactor functions of cobalt corrinoids are a result of their extraordinary organometallic chemistry under physiological conditions and their redox chemistry. Coenzyme B 12 (a reversible source of a free radical), methylcobalamin (a versatile methylating agent), and reduced (supernucleophilic or radicaloid) corrinoids are cofactors in a range of enzymes. These enzymes channel the specific reactivity of B 12 cofactors into biological catalysis of unique reactions, involving radicals and organometallic intermediates. B 12 ‐dependent enzymes thus pose still puzzling structural and mechanistic questions, as does the recently discovered interaction of B 12 cofactors with RNA.

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New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin

Abstract: Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.

Cited by 114 publications

References 41 publications

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

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

Different Biosynthetic Pathways to Fosfomycin in Pseudomonas syringae and Streptomyces Species

Cobalt Enzymes/Models

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