The cleavage of the C-P bond in PRPn to form a-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) is assumed to involve a radical mechanism and PhnJ is a protein that, belonging to the Radical SAM superfamily can perform such a reaction. This enzyme appears to function through the formation of a glycyl radical by the 5'-Ado radical, this radical is then passed onto a conserved cysteine residue. The cysteine radical then forms a covalent bond with the phosphate in the substrate. The glycyl radical is recycled, ready for another reaction to occur.
Kamat SS, Williams HJ, Raushel FM
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.
Kamat SS, Williams HJ, Dangott LJ, Chakrabarti M, Raushel FM
The catalytic mechanism for aerobic formation of methane by bacteria
▸ Abstract
Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms. These bacteria apparently catalyse the formation of methane through the cleavage of the highly unreactive carbon-phosphorus bond in methyl phosphonate (MPn), but the biological or terrestrial source of this compound is unclear. However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate and the bacterial C-P lyase complex is known to convert MPn to methane. In addition to MPn, the bacterial C-P lyase complex catalyses C-P bond cleavage of many alkyl phosphonates when the environmental concentration of phosphate is low. PhnJ from the C-P lyase complex catalyses an unprecedented C-P bond cleavage reaction of ribose-1-phosphonate-5-phosphate to methane and ribose-1,2-cyclic-phosphate-5-phosphate. This reaction requires a redox-active [4Fe-4S]-cluster and S-adenosyl-L-methionine, which is reductively cleaved to L-methionine and 5'-deoxyadenosine. Here we show that PhnJ is a novel radical S-adenosyl-L-methionine enzyme that catalyses C-P bond cleavage through the initial formation of a 5'-deoxyadenosyl radical and two protein-based radicals localized at Gly 32 and Cys 272. During this transformation, the pro-R hydrogen from Gly 32 is transferred to the 5'-deoxyadenosyl radical to form 5'-deoxyadenosine and the pro-S hydrogen is transferred to the radical intermediate that ultimately generates methane. A comprehensive reaction mechanism is proposed for cleavage of the C-P bond by the C-P lyase complex that uses a covalent thiophosphate intermediate for methane and phosphate formation.
