Phosphorus is an essential element for all known forms of life. In living systems, phosphorus is an integral component of nucleic acids, carbohydrates and phospholipids, where it is incorporated as a derivative of phosphate. However, most Gram-negative bacteria have the capability to use phosphonates as a nutritional source of phosphorus under conditions of phosphate starvation1. In these organisms, methylphosphonate is converted to phosphate and methane. In a formal sense, this transformation is a hydrolytic cleavage of a carbon–phosphorus (C–P) bond, but a general enzymatic mechanism for the activation and conversion of alkylphosphonates to phosphate and an alkane has not been elucidated despite much effort for more than two decades. The actual mechanism for C–P bond cleavage is likely to be a radical-based transformation2. In Escherichia coli, the catalytic machinery for the C–P lyase reaction has been localized to the phn gene cluster1. This operon consists of the 14 genes phnC, phnD, …, phnP. Genetic and biochemical experiments have demonstrated that the genes phnG, phnH, …, phnM encode proteins that are essential for the conversion of phosphonates to phosphate and that the proteins encoded by the other genes in the operon have auxiliary functions1,3,4,5,6. There are no functional annotations for any of the seven proteins considered essential for C–P bond cleavage. Here we show that methylphosphonate reacts with MgATP to form α-d-ribose-1-methylphosphonate-5-triphosphate (RPnTP) and adenine. The triphosphate moiety of RPnTP is hydrolysed to pyrophosphate and α-d-ribose-1-methylphosphonate-5-phosphate (PRPn). The C–P bond of PRPn is subsequently cleaved in a radical-based reaction producing α-d-ribose-1,2-cyclic-phosphate-5-phosphate and methane in the presence of S-adenosyl-l-methionine. Substantial quantities of phosphonates are produced worldwide for industrial processes, detergents, herbicides and pharmaceuticals7,8,9. Our elucidation of the chemical steps for the biodegradation of alkylphosphonates shows how these compounds can be metabolized and recycled to phosphate.
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Metcalf, W. W. & Wanner, B. L. Evidence for a fourteen-gene, phnC to phnP locus for phosphonate metabolism in Escherichia coli. Gene 129, 27–32 (1993)
Ahn, Y., Ye, Q., Cho, H., Walsh, C. T. & Floss, H. G. Stereochemistry of carbon-phosphorus cleavage in ethylphosphonate catalyzed by C-P lyase from Escherichia coli. J. Am. Chem. Soc. 114, 7953–7954 (1992)
Metcalf, W. W. & Wanner, B. L. Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation, using TnphoA’ elements. J. Bacteriol. 175, 3430–3442 (1993)
Hove-Jensen, B., Rosenkrantz, T. J., Haldimann, A. & Wanner, B. L. Escherichia coli phnN, encoding ribose 1,5-bisphosphokinase activity (phosphoribosyl diphosphate forming): dual role in phosphonate degradation and NAD biosynthesis pathways. J. Bacteriol. 185, 2793–2801 (2003)
Errey, J. C. & Blanchard, J. S. Functional annotation and kinetic characterization of PhnO from Salmonella enterica. Biochemistry 45, 3033–3039 (2006)
Hove-Jensen, B., McSorley, F. R. & Zechel, D. L. Physiological role of phnP-specified phosphoribosyl cyclic phosphodiesterase in catabolism of organophosphoric acids by the carbon-phosphorus lyase pathway. J. Am. Chem. Soc. 133, 3617–3624 (2011)
Ternan, N. G., McGrath, J. W., McMullan, G. & Quinn, J. P. Organophosphonates: occurrence, synthesis and biodegradation by microorganisms. World J. Microbiol. Biotechnol. 14, 635–647 (1998)
Kononova, S. V. & Nesmeyanova, M. A. Phosphonates and their degradation by microorganisms. Biochemistry (Mosc.) 67, 184–195 (2002)
White, A. K. & Metcalf, W. W. Microbial metabolism of reduced phosphorus compounds. Annu. Rev. Microbiol. 61, 379–400 (2007)
Avila, L. Z., Draths, K. M. & Frost, J. W. Metabolites associated with organophosphonate C-P bond cleavage: chemical synthesis and microbial degradation of [32P]-ethylphosphonic acid. Bioorg. Med. Chem. Lett. 1, 51–54 (1991)
Frost, J. W., Loo, S., Cordeiro, M. L. & Li, D. Radical-based dephosphorylation and organophosphonate biodegradation. J. Am. Chem. Soc. 109, 2166–2171 (1987)
Parker, G. F., Higgins, T. P., Hawkes, T. & Robson, R. L. Rhizobium (Sinorhizobium) meliloti phn genes: characterization and identification of their protein products. J. Bacteriol. 181, 389–395 (1999)
Kamat, S. S. et al. Catalytic mechanism and three-dimensional structure of adenine deaminase. Biochemistry 50, 1917–1927 (2011)
Maynes, J. T., Yuan, R. G. & Snyder, F. F. Identification, expression and characterization of the Escherichia coli guanine deaminase. J. Bacteriol. 182, 4658–4660 (2000)
Hall, R. S. et al. Three dimensional structure and catalytic mechanism of cytosine deaminase. Biochemistry 50, 5077–5085 (2011)
Krenitsky, T. A., Neil, S. M., Elion, G. B. & Hitchings, G. H. A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Arch. Biochem. Biophys. 150, 585–599 (1972)
La . Vallie, E. R., McCoy, J. M., Smith, D. B. & Riggs, P . Enzymatic and chemical cleavage of fusion proteins. Curr. Protocols Mol. Biol. Unit 16.4B. (1994)
Jochimsen, B. et al. Five phosphonate operon gene products as components of a multi-enzyme complex of the carbon-phosphorus lyase pathway. Proc. Natl Acad. Sci. USA 108, 11393–11398 (2011)
Seibert, C. M. &. Raushel, F. M. Structural and catalytic diversity within the amidohydrolase superfamily. Biochemistry 44, 6383–6391 (2005)
Frey, P. A., Hegeman, A. D. & Ruzicka, F. J. The radical SAM superfamily. Crit. Rev. Biochem. Mol. Biol. 43, 63–88 (2008)
Chatterjee, A. et al. Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nature Chem. Biol. 4, 758–765 (2008)
McGlynn, S. E. et al. Identification and characterization of a novel member of the radical AdoMet enzyme superfamily and implications for the biosynthesis of the Hmd hydrogenase active site cofactor. J. Bacteriol. 192, 595–598 (2010)
Zhang, Y. et al. Diphthamide biosynthesis requires an organic radical generated by iron-sulphur enzyme. Nature 465, 891–896 (2010)
Parast, C. V., Wong, K. K., Lewisch, S. A. & Kozarich, J. W. Hydrogen exchange of the glycyl radical of pyruvate formate-lyase is catalyzed by cysteine 419. Biochemistry 34, 2393–2399 (1995)
Buis, J. M. & Broderick, J. B. Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation. Arch. Biochem. Biophys. 433, 288–296 (2005)
Thauer, R. K. & Shima, S. Methane as fuel for anaerobic microorganisms. Ann. NY Acad. Sci. 1125, 158–170 (2008)
We thank D. Barondeau and his laboratory for use of the anaerobic chamber and for advice on the assembly of Fe–S clusters in radical SAM enzymes. We also thank C. Xu for help with some of the 31P NMR spectra and S. Burrows for help with cloning phnI and phnH. This work was supported in part by the National Institutes of Health (GM93342, GM71790) and the Robert A. Welch Foundation (A-840). The cryoprobe for the NMR spectrometer was purchased with funds from the National Science Foundation (0840464)
The authors declare no competing financial interests.
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Kamat, S., Williams, H. & Raushel, F. Intermediates in the transformation of phosphonates to phosphate by bacteria. Nature 480, 570–573 (2011). https://doi.org/10.1038/nature10622
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