FIG 3.
HPLC traces of reaction mixtures. Five-hundred-microliter samples were loaded onto a C18 column and eluted with a gradient of 50 mM phosphate buffer (pH 5.3)-acetonitrile mixture (percent acetonitrile is indicated by the dashed lines). CoA esters were detected by absorbance at 254 nm. (A) Androst-4-ene-3,17-dione standard (100 μM). (B) Propionyl-CoA standard (100 μM). (C to E) Reaction mixtures containing 100 μM 3-OPC-CoA and 200 μM ferrocenium hexafluorophosphate in 100 mM TAPS buffer (pH 8.5), with the addition of 0.5 μM (each) FadE28-FadE29 and ChsH1-ChsH2-Ltp2 (C), 0.5 μM (each) FadE28-FadE29 and ChsH1-ChsH2 (D), or 0.5 μM FadE28-FadE29 (E). In the presence of Ltp2, peak III, corresponding to the retention times of 3-OPDC-CoA and 17-HOPC-CoA in chromatograms D and E, was reduced and two new peaks (I and II) appeared, corresponding to the retention times of propionyl-CoA and androst-4-ene-3,17-dione, respectively.