Involvement of NADPH in the cyclization reaction of carotenoid biosynthesis

Abstract

Cyclic carotenoids, e.g. L-carotene, are formed by cyclization of an acyclic precursor, lycopene. The gene, crtY, which encodes lycopene L-cyclase, has a partial sequence characteristic of a pyridine nucleotide binding domain, and NAD(P)H has been reported to be an absolute requirement for the cyclization reaction in vitro. By complementary incubations with lycopene as substrate and with (4R)-[4-2H]NADPH in 1H2O or with unlabelled NADPH in 2H2O in the presence of the purified enzyme, it has now been shown that the hydrogen atom introduced at C(2) in the cyclization comes from water and not from NADPH. The previously proposed mechanism involving the initiation of cyclization by H+ attack at C(2) of the folded acyclic end group of the precursor is thus confirmed. No hydrogen is transferred from NADPH, which is therefore not involved directly in the cyclization reaction, but must play an indirect role, e.g. as an allosteric activator. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.