Kinetic characterisation of the light-driven protochlorophyllide oxidoreductase (POR) from Thermosynechococcus elongatus.
The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of the C17-C18 double bond of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), which is a key regulatory step in the chlorophyll biosynthesis pathway. POR from the thermophilic cyanobacterium Thermosynechococcus elongatus is an attractive system for following the reaction and in the present work we have carried out a detailed steady state kinetic characterisation of this enzyme. The thermophilic POR was shown to have maximal activity at approximately 50 degrees C, which is similar to the growth temperature of the organism. The V(max) was calculated to be 0.53 microM min(-1) and the K(m) values for NADPH and Pchlide were 0.013 microM and 1.8 microM, respectively. The binding properties for both substrates as well as the NADP(+) product have been analysed by using fluorescence emission measurements, which have allowed the dissociation constants for binding to be calculated. These results represent the first steady state kinetic characterisation of a thermophilic version of POR.
McFarlane
Michael J
MJ
Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, United Kingdom.
Hunter
C Neil
CN
Heyes
Derren J
DJ
D002384:Catalysis / Q000528:radiation effects; D002735:Chlorophyllides / Q000737:chemistry / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology*; D004795:Enzyme Stability; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008027:Light; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002735:Chlorophyllides; D009249:NADP; D006859:Hydrogen; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase