Intermediates in the transformation of phosphonates to phosphate by bacteria

Abstract

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 catalyzed by PhnI, PhnM, and PhnJ

a, RPnTP from the reaction of methyl phosphonate and MgATP catalyzed by PhnI in the presence of PhnG, PhnH, and PhnL at pH 8.5. The inset shows the ³¹P-³¹P coupling of the triphosphate portion of the RPnTP product. b, The formation of PRPn from RPnTP in the presence of PhnM. The inset displays the proton coupled spectrum at pH 8.5 showing the multiplet that corresponds to the 1-phosphonylmethane and the triplet that corresponds to the 5-phosphate. c, The formation of PRcP from PRPn in the presence of PhnJ at pH 6.8. The inset displays the proton coupled spectrum showing the formation of a new triplet that corresponds to the 1,2-cyclic moiety of PRcP. The chemical shifts for the phosphate moiety at C5 of PRcP and PRPn are coincident with one another (3.4 ppm).

Figure 2. UV-visible absorbance spectrum of PhnJ (31 μM) after anaerobic reconstitution of the iron-sulfur cluster

The peak at 280 nm for the solid line is due to the protein and the absorbance centered 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]¹⁺ 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 likely initiated via an electron transfer from the iron-sulfur cluster to reductively cleave SAM to transiently generate L-methionine and a 5′-deoxyadenosyl radical. The 5′-deoxyadenosyl radical may subsequently catalyze the formation of a protein radical, presumably a cysteine-based thiyl radical. Thiyl radicals have previously been demonstrated in pyruvate-formate lyase^, and methyl-coenzyme M reductase. 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 C2 hydroxyl of the substrate would generate PRcP and the free thiol group. Methane would be formed via hydrogen atom abstraction from either 5-deoxyadenosine or the putative cysteine residue.

Figure 4. Reaction pathway for the conversion of methyl phosphonate to PRcP

The proteins PhnG, PhnH, PhnI, PhnJ, PhnL, and PhnM are required for this transformation. The role of PhnK is unknown.