FIG 3.

Identification of ydcJ (PP_5260) as a 2OA decarboxylase/hydroxylase. (A) Growth of the wild-type strain (black) and the PP_5260 mutant (red) on d-lysine (line) or l-lysine (dashed line) as a sole carbon source. The shaded area represents 95% CI; n = 3. (B) HPLC traces representing results of in vitro reactions run with apo PP_5260 with exogenous metals added at 50 μM. Retention times for 2OA and 2HG are shown by vertical dashed lines. Metal or EDTA control is indicated to the right of traces. (C) In vitro assay of 2OA conversion to 2HG by purified PP_5260 protein analyzed via the LC-TOF method. 2OG in white, 2HG in black. (D) In vitro assay of purified PP_5260 protein with 2OA as the substrate. The black bar represents the concentration of d-2HG measured by enzyme coupled assay. The white bar represents the total 2HG concentration as measured by the LC-TOF method. Error bars represent 95% CI; n = 3. (E) Initial velocity of reaction catalyzed by PP_5260 as a function of 2OA concentration. Blue dots represent individual measurements, while the black fit line represents a Michaelis-Menten fit. (F) Chemical reaction catalyzed by PP_5260; 2OA is decarboxylated to d-2HG.