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.