Fig. 1.

AtCAO displays Chlide a oxygenase activity in vitro. (A) SDS/PAGE of ³⁵S-AtCAO (lane a) and lack of ³⁵S-AtCAO in wheat germ extracts programmed with the naked vector DNA missing the AtCAO cDNA insert (lane b). (B) Chlide a oxygenase activity of AtCAO (dotted line) and of the respective control without AtCAO (solid line). The curves show HPLC tracings of acetone-extracted pigments at 435 nm using a photodiode array detector. The pigment eluting at 19.5 min corresponds to Chlide a, whereas the pigment eluting at 16.25 min was identified as Chlide b by absorbance measurements (Inset). (C) Confirmation of the identity of Chlide b eluting at 16.25 min by matrix-assisted laser desorption/ionization mass spectroscopy. The determined molecular mass (630.7 kDa) exactly matched the theoretical isotopic distribution of C35H32N4O6Mg corresponding to Chlide b. The matrix used was terthiophene (molecular mass of 248.4 Da).