Extended Data Fig. 6: Reaction of NMO Orf1480 with carrier protein-tethered nitronates.

a, Scheme of enzymatic reactions and chemical derivatization using DNPH. The nitroacetyl-S-carrier protein was generated from EtmA or UnkA reactions with unlabeled or labeled Gly as the substrate, as described for each panel. b,c, LC-MS analysis of derivatized mixtures in negative ion mode. b, Extracted-ion chromatogram (EIC) from LC-MS analysis, with m/z 198, 253 and 254 extracted, corresponding to DNPH, 15 and 15’, respectively. I) Derivatization of a glyoxylate chemical standard; II) Derivatization of the reaction with unlabeled Gly as the substrate; III) Derivatization of the reaction with 2-¹³C-¹⁵N-Gly as the substrate. c, MS signals displayed by each peak. I) Derivatization of the reaction with unlabeled Gly as the substrate; II) Derivatization of the reaction with 2-¹³C-¹⁵N-Gly as the substrate. Two peaks, consistent with DNPH-derivatized glyoxylate 15, were observed, which are assigned as isomers of carbon-nitrogen double bond (E-/Z-15), although which peak is E- or Z-form is unknown. As expected, comparing with the results of unlabeled-Gly added mixture, we detected +1 Da peak in the mixture of 2-¹³C-¹⁵N-Gly added, which is consistent with loss of one nitrogen and confirmed the formation of nitronate.