The catalytic mechanism for aerobic formation of methane by bacteria

A mechanism is proposed for the formation of methane by bacteria, through the cleavage of a highly unreactive carbon–phosphorus bond in methyl phosphonate by PhnJ in the bacterial C–P lyase complex. Novel bacterial biosynthesis of methane Aerobic marine organisms produce significant quantities of the potent greenhouse gas methane, much of it via the cleavage of the highly unreactive carbon–phosphorus bonds of alkylphosphonates. In this study the authors explore the mechanism of PhnJ, an unusual radical S -adenosyl- L -methionine (SAM) enzyme that appears to use a cysteine-based thiyl radical to help catalyse the conversion of the alkylphosphonate substrate to methane and ribose-1,2-cyclic phosphate-5-phosphate. This reaction, not previously encountered in biological chemistry, establishes a novel mechanism for cleaving carbon–phosphorus bonds to form methane and phosphate via a covalent thiophosphate intermediate. Methane is a potent greenhouse gas that is produced in significant quantities by aerobic marine organisms 1 . 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 2 . However, the ocean-dwelling bacterium Nitrosopumilus maritimus catalyses the biosynthesis of MPn from 2-hydroxyethyl phosphonate 3 and the bacterial C–P lyase complex is known to convert MPn to methane 4 , 5 , 6 , 7 . 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 4 , 5 , 6 , 7 . 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 8 . 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 mecha