Purification and kinetic analysis of pea (Pisum sativum L.) NADPH:protochlorophyllide oxidoreductase expressed as a fusion with maltose-binding protein in Escherichia coli.
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.
Martin
G E
GE
Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
Timko
M P
MP
Wilks
H M
HM
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins / Q000096:biosynthesis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010088:Oxidoreductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D018532:Pisum sativum / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase