Catalytic properties of the expressed acyclic carotenoid 2-ketolases from Rhodobacter capsulatus and Rubrivivax gelatinosus.
Purple photosynthetic bacteria synthesize the acyclic carotenoids spheroidene and spirilloxanthin which are ketolated to spheroidenone and 2,2'-diketospirilloxanthin under aerobic growth. For the studies of the catalytic reaction of the ketolating enzyme, the crtA genes from Rubrivivax gelatinosus and Rhodobacter capsulatus encoding acyclic carotenoid 2-ketolases were expressed in Escherichia coli to functional enzymes. With the purified enzyme from the latter, the requirement of molecular oxygen and reduced ferredoxin for the catalytic activity was determined. Furthermore, the putative intermediate 2-HO-spheroidene was in vitro converted to the corresponding 2-keto product. Therefore, a monooxygenase mechanism involving two consecutive hydroxylation steps at C-2 were proposed for this enzyme. By functional pathway complementation studies in E. coli and enzyme kinetic studies, the product specificity of both enzymes were investigated. It appears that the ketolases could catalyze most intermediates and products of the spheroidene and spirilloxanthin pathway. This was also the case for the enzyme from Rba. capsulatus from which spirilloxanthin synthesis is absent. In general, the ketolase of Rvi. gelatinosus had a better specificity for spheroidene, HO-spheroidene and spirilloxanthin as substrates than the ketolase from Rba. capsulatus.
Gerjets
Tanja
T
Biosynthesis Group, Molecular Biosciences 213, J. W. Goethe Universität, P.O. Box 111932, D-60054 Frankfurt, Germany.
Steiger
Sabine
S
Sandmann
Gerhard
G
D020562:Betaproteobacteria / Q000201:enzymology*; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013379:Substrate Specificity; D024341:Xanthophylls / Q000737:chemistry / Q000378:metabolism
D024341:Xanthophylls; C005637:spheroidene; C104880:spheroidenone; D002338:Carotenoids; D006836:Hydro-Lyases; C475147:carotenoid 1,2-hydratase; C055255:spirilloxanthin