In vivo and in vitro studies on the carotenoid cleavage oxygenases from Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1 revealed their substrate specificities and non-retinal-forming cleavage activities.
Carotenoid cleavage oxygenases are nonheme iron enzymes that specifically cleave carbon-carbon double bonds of carotenoids. Their apocarotenoid cleavage products serve as important signaling molecules that are involved in various biological processes. A database search revealed the presence of putative carotenoid cleavage oxygenase genes in the genomes of Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1. The four genes sala_1698, sala_1008, ppsir1_15490 and ppsir1_17230 were cloned and heterologously expressed in carotenoid-producing Escherichia coli JM109 strains. Two of the four encoded proteins exhibited carotenoid cleavage activity. S. alaskensis RB2256 carotenoid cleavage oxygenase (SaCCO), which is encoded by sala_1698, was shown to cleave acyclic and monocyclic substrates. Coexpression of sala_1698 in carotenoid-producing E. coli JM109 strains revealed cleavage activity for lycopene, hydroxylycopene, and dihydroxylycopene. The monocyclic substrate apo-8'-carotenal was cleaved in vitro by purified SaCCO at the 9'/10' and 11'/12' double bonds. The second enzyme, P. pacifica SIR-1 carotenoid cleavage oxygenase (PpCCO), is encoded by ppsir1_15490. PpCCO-mediated carotenoid cleavage requires the presence of either hydroxy or keto groups. PpCCO cleaved zeaxanthin, hydroxylycopene, and dihydroxylycopene, and also the C(50) carotenoids decaprenoxanthin, sarprenoxanthin and sarcinaxanthin, in carotenoid-producing E. coli JM109 strains. Whole cells of E. coli JM109 overexpressing ppsir1_15490mut, a mutant of ppsir1_15490 with enhanced gene expression, were applied for the conversion of carotenoids. Analysis of the carotenoid cleavage products revealed a single cleavage site at the 13'/14' double bond for astaxanthin, and two cleavage sites at the 11'/12' or 13'/14' double bond for zeaxanthin, nostoxanthin, and canthaxanthin.
Institute of Industrial Genetics, University of Stuttgart, Stuttgart, Germany.
Jana
J
Hoffmann
Judit
J
Bóna-Lovász
Holger
H
Beuttler
Josef
J
Altenbuchner
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D000077276:Lycopene; D013058:Mass Spectrometry; D015394:Molecular Structure; D009154:Mutation; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D020560:Proteobacteria / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D012176:Retinoids / Q000737:chemistry / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D024341:Xanthophylls / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D007527:Isoenzymes; D012176:Retinoids; D024341:Xanthophylls; C014802:sarcinaxanthin; C011431:decaprenoxanthin; D002338:Carotenoids; D010105:Oxygenases; C500451:carotenoid oxygenase; D000077276:Lycopene