title: Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay.
abstract: An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible.
authors.affiliation: Central Laboratories for Key Technology, Kirin Brewery Co., Ltd, Kanagawa, Japan.
authors.forename: P D
authors.initials: PD
authors.lastname: Fraser
authors.forename: H
authors.initials: H
authors.lastname: Shimada
authors.forename: N
authors.initials: N
authors.lastname: Misawa
mesh_terms: D000421:Alcaligenes / Q000201:enzymology; D001419:Bacteria / Q000201:enzymology; D002338:Carotenoids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D008055:Lipids / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000378:metabolism; D015394:Molecular Structure; D010105:Oxygenases / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology; D024341:Xanthophylls; D019207:beta Carotene / Q000031:analogs & derivatives* / Q000096:biosynthesis
chemical_list: D003902:Detergents; D008055:Lipids; D024341:Xanthophylls; D019207:beta Carotene; D002338:Carotenoids; C005948:astaxanthine; D006899:Mixed Function Oxygenases; D010105:Oxygenases