NADPH:protochlorophyllide oxidoreductase from Synechocystis: overexpression, purification and preliminary characterisation.
NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexahistidine tag at the N-terminus. This enzyme (His(6)-POR) has been purified to homogeneity and a preliminary characterisation of its kinetic and substrate binding properties is presented. Chemical modification experiments have been used to demonstrate inhibition of POR activity by the thiol-specific reagent N-ethyl maleimide. Substrate protection experiments reveal that the modified Cys residues are involved in either substrate binding or catalysis.
Heyes
D J
DJ
Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, The University of Sheffield, S10 2TN, Sheffield, UK.
Martin
G E
GE
Reid
R J
RJ
Hunter
C N
CN
Wilks
H M
HM
D002735:Chlorophyllides / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011485:Protein Binding; D011521:Protochlorophyllide / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D002735:Chlorophyllides; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D011521:Protochlorophyllide; D006639:Histidine; D009249:NADP; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase; D005033:Ethylmaleimide