Abstract
NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key reaction in the chlorophyll biosynthetic pathway. To facilitate structure-function studies, POR from pea (Pisum sativum L.) has been overexpressed in Escherichia coli as a fusion with maltose-binding protein (MBP) at 5-10% of the total soluble cell protein. The fusion protein (MBP-POR) has been purified to greater than 90% homogeneity by a two-step affinity-purification procedure. This represents the first successful overexpression and purification of a plant POR. MBP-POR was found to be active, and the kinetic properties were determined using a continuous assay in which the rate of chlorophyllide formation was measured. The Vmax was 20.6+/-0.9 nmol.min-1.mg-1 and the Km values for NADPH and protochlorophyllide were 8.7+/-1.9 microM and 0.27+/-0.04 microM respectively. These results represent the first determination of the kinetic properties of a pure POR and the first report on the kinetics of POR from a dicotyledenous plant. The experiments described here demonstrate that the enzyme is not a 'suicide' enzyme, and the only components required for catalysis are NADPH, protochlorophyllide and light. Size-exclusion chromatography on a Superose 6 HR column indicated that MBP-POR has a molecular mass of 155 kDa (compared with the molecular mass of 80 kDa estimated by SDS/PAGE), indicating that it behaves as a dimer in solution. This is the first direct determination of the oligomerization state of POR.