Phosphonates to Phosphate: A Functional Annotation of the Essential Genes of the Phn Operon in Escherichia coli

The reaction mechanism for the enzymatic conversion of methyl phosphonate to phosphate and methane in Escherichia coli has eluded researchers over the last three decades despite significant genetic and in vivo studies. The phn operon governs the C-P lyase activity in E. coli. The essential genes within the phn operon are phnGHIJKLM. The proteins encoded by phnGHM were over-expressed in E. coli and purified to homogeneity using standard protocols. The proteins encoded by phnIJKL were soluble only when expressed as N-terminal glutathione S-transferase (GST) fusion proteins. PhnI was shown to catalyse the formation of a-d-ribose-1-methylphosphonate-5-triphosphate (RPnTP) from MgATP and methylphosphonate in the presence of PhnG, PhnH, and PhnL after in situ cleavage of the GST-tags. PhnI alone catalyses the hydrolytic cleavage of MgATP to adenine and d-ribose-5-triphosphate (RTP). PhnM catalyses the hydrolysis of a-d-ribose-1-methylphosphonate-5-triphosphate (RPnTP) to a-d-ribose-1-methylphosphonate-5-phosphate (PRPn) and pyrophosphate with attack of water on the a-phosphoryl group of the triphosphate moiety of RPnTP. PhnJ was reconstituted with an iron-sulphur cluster through the anaerobic addition of FeSO4, Na2S and Na2S2O4 under strictly anaerobic conditions. The [Fe4S4]-reconstituted PhnJ 1 http://www.beilstein-institut.de/escec2011/Proceedings/Raushel/Raushel.pdf Experimental Standard Conditions of Enzyme Characterization September 12 – 16, 2011, Rüdesheim/Rhein, Germany