XANTHOPHYLL BIOSYNTHESIS - MOLECULAR AND FUNCTIONAL-CHARACTERIZATION OF CAROTENOID HYDROXYLASES FROM PEPPER FRUITS (CAPSICUM-ANNUUM L.)

To dissect the mechanism by which carotenoid hydroxylases catalyze xan thophyll formation, we have cloned two pepper cDNAs encoding beta-cryp toxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro syste m, we find that both enzymes are ferredoxin dependent and that their a ctivity is strongly inhibited by iron chelators such as o-phenanthroli ne or 8-hydroxyquinoline. This suggests the transfer of a reducing equ ivalent from NADPH to the hydroxylase via ferredoxin and the involveme nt of an iron activated oxygen insertion process. Based on sequence an alysis, the putative histidine clusters involved in the iron coordinat ion were identified and their roles evaluated. Following site-directed mutagenesis of the identified histidine residues hydroxylase activity was totally inactivated. Collectively, our data indicate that caroten oid hydroxylases belong to a new class of diiron proteins structurally related to membrane fatty acid desaturases. Mechanistically, both typ es of enzymes exploit iron activated oxygen to break the C-H bond with concomitant formation of double bond or oxygen insertion. We propose that the same mechanism operates for beta-carotene ketolase and probab ly for other carotenoid oxygenases as well. (C) 1998 Elsevier Science B.V.