Figure 3. YydG catalyzes amino acid epimerization.

LC-MS/MS analysis of (a) L-/D-Leu and L-Ile/D-allo-Ile or (b) L-/D-Val. Upper traces were obtained with standard amino acids while lower traces correspond to amino acids obtained after incubation of YydF_18-49 with the radical SAM enzyme YydG in deuterated buffer. Amino acids were analyzed after hydrolysis and derivatization by N-α-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA). MS spectra of (c) L-Ile-FDVA, (d) L-Val-FDVA (upper traces) and their D-epimers (lower traces) obtained after incubation of YydF_18-49 with YydG in deuterated buffer. Mass spectra correspond to the dominant ions fragments: m/z= 366 and 352 for Ile- and Val-FDVA derivatives, respectively. The amino acids were derivatized by L-FDVA and detected by LC-MS after ion current extraction in MS/MS experiments using the transition 412>366 and 398>352 for Ile/Leu and Val-FDVA derivatives, respectively. (e) LC-MS/MS analysis of the deuterated peptide YydF_18-49-VD₈ incubated with YydG. After incubation with YydG, YydF_18-49-VD₈ ([M+2H]²⁺ = 1887.7) (trace 1) was converted in peptides containing one D-valine ([M+2H]²⁺ = 1887.2) (traces 2&3) or two D-Val [M+2H]²⁺ = 1886.7 (trace 4). In the sequences, the octadeuterated L-valine residues are labeled in light blue and the heptadeuterated D-valine in red. Masses indicate mono-isotopic ions. (f) LC-MS/MS analysis of 5'-dA produced by YydG incubated with YydF_18-49 (upper trace) or YydF_18-49-VD₈ (lower trace). (g) Time-course production of 5'-dA (blue symbol) and epimerized peptide (red symbol). See supplementary Information for experimental conditions.