Fig. 1.

In vitro assay of MpnS activity. (A) Crude cell extract from an E. coli MpnS overexpression strain was incubated aerobically with 1-¹³C-HEP in the presence of Fe(II) and the phosphorus-containing products were examined using ³¹P NMR spectroscopy. After incubation for 1 hour a single product was observed as a doublet centered at 23.5 ppm. The mass and retention time of this product determined by LC-MS is consistent with this product being 1-¹³CMPn (Fig. S3). (B) Spiking of this reaction with the substrate, 1-¹³C-HEP produced a second doublet centered at 19 ppm, showing that the substrate was completely consumed in the initial reaction. (C) The identity of the reaction products was determined using ¹³C NMR after repeating the assay in a sealed vial using purified MpnS with a mixture of 1-¹³C-HEP and 2-¹³C-HEP as substrates. The C-2 labeled carbon of HEP is converted to ¹³C bicarbonate (H¹³CO ^-₃), while the C-1-labelled carbon is converted to 1-¹³C-MPn. Bonding to phosphorus splits the ¹³C peak in the NMR spectrum. Thus, the C-1 peak is split and the C-2 peak is not. Glycerol, a component of the assay mixture, is also observed in the ¹³C spectrum. The ¹³C label is indicated by a red asterisk.