Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay.
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.
Fraser
P D
PD
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd, Kanagawa, Japan.
Shimada
H
H
Misawa
N
N
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
D003902:Detergents; D008055:Lipids; D024341:Xanthophylls; D019207:beta Carotene; D002338:Carotenoids; C005948:astaxanthine; D006899:Mixed Function Oxygenases; D010105:Oxygenases