Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts--partial purification and biochemical properties.
Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersion esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa. with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with Km values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 microM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including beta-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences.
Fraser
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PD
Division of Biochemistry, School of Biological Sciences, University of London, Egham, Surrey, UK.
Schuch
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W
Bramley
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PM
D000255:Adenosine Triphosphate / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D000255:Adenosine Triphosphate; D019883:Alkyl and Aryl Transferases; D051232:Geranylgeranyl-Diphosphate Geranylgeranyltransferase; D008274:Magnesium