Figure 5

Inhibition of BCMO1 by diphenylamine. A) Inhibition of BCMO1 by diphenylamine in vitro. Diphenylamine (50-1000 μM) was added from a 20 mM stock in DMSO to an in vitro reaction mixture prior to addition of substrate. The final concentration of DMSO in all samples was 5%. The enzyme was preincubated with the inhibitor for 30 min at 4°C in the standard reaction buffer and was then added to 20 μM β-carotene and activity determined as described in Methods. A representative experiment is shown. B) Inhibition of BCMO1 by diphenylamine in vivo. E. coli cultures transformed with both the plasmid for β-carotene synthesis and the BCMO1 plasmid were grown overnight (12-16 hr) to allow accumulation of β-carotene. At this point, the inhibitor diphenylamine (at a final concentration of 200 μM from a 200 mM stock solution in MeOH) was added to prevent further synthesis of carotenoids [38]. The cultures were shaken for 2 hours, split in half, and one half was induced by the addition of arabinose (0.02% final concentration). Both cultures were shaken for an additional 7 hours and analyzed by the in vivo assay as described in Methods (N = 3).