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An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis.

Cicchillo RM, Zhang H, Blodgett JA, Whitteck JT, Li G, Nair SK, van der Donk WA, Metcalf WW - Nature (2009)

Bottom Line: Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
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.

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NMR and mass spectral data from in vitro labelling studiesa,31P NMR spectrum showing production of 13C-HMP (δ 17.2 ppm) from 1-13C-HEP (δ 19.9 ppm) after 50% conversion. Inset: mass spectrum of HMP derived from a reaction utilizing 1,1-2H2-HEP. b, Mass spectrum of 18O,13C-formate (m/z 106) produced by HEPD from a reaction utilizing 18O2 and 2-13C-HEP. Spurious formate is denoted by an asterisk (m/z 103). c, Analysis of HMP produced from the HEPD reaction performed with 18O2. Extracted ion chromatograms demonstrate ∼60 % of the HMP contains 18O (black line) while 40 % contains 16O (red line). Inset: summed mass spectrum from the total ion chromatographic peak. d, Summary of labelling studies from the HEPD reaction.
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Figure 2: NMR and mass spectral data from in vitro labelling studiesa,31P NMR spectrum showing production of 13C-HMP (δ 17.2 ppm) from 1-13C-HEP (δ 19.9 ppm) after 50% conversion. Inset: mass spectrum of HMP derived from a reaction utilizing 1,1-2H2-HEP. b, Mass spectrum of 18O,13C-formate (m/z 106) produced by HEPD from a reaction utilizing 18O2 and 2-13C-HEP. Spurious formate is denoted by an asterisk (m/z 103). c, Analysis of HMP produced from the HEPD reaction performed with 18O2. Extracted ion chromatograms demonstrate ∼60 % of the HMP contains 18O (black line) while 40 % contains 16O (red line). Inset: summed mass spectrum from the total ion chromatographic peak. d, Summary of labelling studies from the HEPD reaction.

Mentions: The fate of the excised carbon was determined using synthetic 2-13C-HEP. Stoichiometric amounts of HMP and a new product with a resonance of 171 ppm in the 13C NMR spectrum were observed, corresponding to 13C-formate (Supplementary Fig. 1). Additionally, HMP formed with 1-13C-HEP and 2-13C-HEP was analysed by 31P NMR spectroscopy, resulting in a doublet (from 13C-HMP, Fig. 2a) and a singlet signal (from 12C-HMP), respectively. The hydrogens at C1 are not removed in this process as the HEPD-catalysed oxidation of 1-2H2-HEP produced 1-2H2-HMP (Fig. 2a, inset).


An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis.

Cicchillo RM, Zhang H, Blodgett JA, Whitteck JT, Li G, Nair SK, van der Donk WA, Metcalf WW - Nature (2009)

NMR and mass spectral data from in vitro labelling studiesa,31P NMR spectrum showing production of 13C-HMP (δ 17.2 ppm) from 1-13C-HEP (δ 19.9 ppm) after 50% conversion. Inset: mass spectrum of HMP derived from a reaction utilizing 1,1-2H2-HEP. b, Mass spectrum of 18O,13C-formate (m/z 106) produced by HEPD from a reaction utilizing 18O2 and 2-13C-HEP. Spurious formate is denoted by an asterisk (m/z 103). c, Analysis of HMP produced from the HEPD reaction performed with 18O2. Extracted ion chromatograms demonstrate ∼60 % of the HMP contains 18O (black line) while 40 % contains 16O (red line). Inset: summed mass spectrum from the total ion chromatographic peak. d, Summary of labelling studies from the HEPD reaction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874955&req=5

Figure 2: NMR and mass spectral data from in vitro labelling studiesa,31P NMR spectrum showing production of 13C-HMP (δ 17.2 ppm) from 1-13C-HEP (δ 19.9 ppm) after 50% conversion. Inset: mass spectrum of HMP derived from a reaction utilizing 1,1-2H2-HEP. b, Mass spectrum of 18O,13C-formate (m/z 106) produced by HEPD from a reaction utilizing 18O2 and 2-13C-HEP. Spurious formate is denoted by an asterisk (m/z 103). c, Analysis of HMP produced from the HEPD reaction performed with 18O2. Extracted ion chromatograms demonstrate ∼60 % of the HMP contains 18O (black line) while 40 % contains 16O (red line). Inset: summed mass spectrum from the total ion chromatographic peak. d, Summary of labelling studies from the HEPD reaction.
Mentions: The fate of the excised carbon was determined using synthetic 2-13C-HEP. Stoichiometric amounts of HMP and a new product with a resonance of 171 ppm in the 13C NMR spectrum were observed, corresponding to 13C-formate (Supplementary Fig. 1). Additionally, HMP formed with 1-13C-HEP and 2-13C-HEP was analysed by 31P NMR spectroscopy, resulting in a doublet (from 13C-HMP, Fig. 2a) and a singlet signal (from 12C-HMP), respectively. The hydrogens at C1 are not removed in this process as the HEPD-catalysed oxidation of 1-2H2-HEP produced 1-2H2-HMP (Fig. 2a, inset).

Bottom Line: Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus