Phosphonate degradation in E. coli has been a long standing mystery. The EFI’s AH Bridging Project successfully identified probable nucleosidase, hydrolase (an AH target), and radical SAM enzymes within the phosphonate operon of E. coli that collectively convert methyl phosphonate to methane and ribose-1,2-cylic-phosphate-5-phosphate. This work shows how enzyme superfamilies can be leveraged to discover novel biology.
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 starvation. 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 transformation. In Escherichia coli, the catalytic machinery for the C-P lyase reaction has been localized to the phn gene cluster. 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 functions. 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 pharmaceuticals. Our elucidation of the chemical steps for the biodegradation of alkylphosphonates shows how these compounds can be metabolized and recycled to phosphate.
Figure 1. 31P NMR spectra of the reaction products catalysed by PhnI, PhnM and PhnJ. a, RPnTP from the reaction of methylphosphonate and MgATP catalysed by PhnI in the presence of PhnG, PhnH, and PhnL at pH 8.5. The methylphosphonyl group is labelled as 1-Pn. Inset, 31P–31P coupling of the triphosphate portion of the RPnTP product (the α, β and γ phosphoryl groups). b, The formation of PRPn from RPnTP in the presence of PhnM. Inset, proton-coupled spectrum at pH 8.5 showing the multiplet that corresponds to 1-Pn and the triplet that corresponds to the 5-phosphate (5P). Pi, inorganic phosphate. c, The formation of PRcP from PRPn in the presence of PhnJ at pH 6.8. Inset, proton-coupled spectrum showing the formation of a new triplet that corresponds to the 1,2-cyclic moiety of PRcP (1,2-cP). The chemical shifts for the phosphate moiety at the fifth carbon atom of PRcP and PRPn are coincident with one another (3.4 p.p.m.).
Figure 2: Ultraviolet–visible absorbance spectrum of PhnJ (31 μM) after anaerobic reconstitution of the Fe–S cluster (solid line). The peak at 280 nm is due to the protein and the absorbance centred at 403 nm represents the [4Fe–4S]2+ cluster. The dotted line represents the absorbance spectrum of PhnJ (27 μM) reconstituted with a [4Fe–4S]2+ cluster after reduction of the cluster with sodium dithionite to the [4Fe–4S]+ form.
Figure 3: Working model for the transformation of PRPn to PRcP. The cleavage of the C–P bond in PRPn by PhnJ reconstituted with a [4Fe–4S]1+ cluster and SAM is probably initiated by electron transfer from the Fe–S cluster to reductively cleave SAM and thus transiently generate L-methionine (L-met) and a 5′-deoxyadenosyl radical (Ado- H2). This radical may subsequently catalyse the formation of a protein radical (PhnJ-X•), presumably a cysteine-based thiyl radical. Thiyl radicals have previously been demonstrated in pyruvate formate lyase24, 25 and methyl coenzyme M reductase26. The thiyl radical may attack the phosphonate moiety of the substrate to liberate a methyl radical with formation of a thioester intermediate. Intramolecular attack by the hydroxyl of the second carbon atom of the substrate would generate PRcP and the free thiol group. Methane would be formed through hydrogen atom abstraction from either 5-deoxyadenosine or the putative cysteine residue.
Reprinted with permission from Macmillan Publishers Ltd: Nature, © 2011.