Molecular Analysis of Carotenoid Cyclase Inhibition

Later steps of carotenoid biosynthesis catalyzed by cyclase enzymes involve the formation of α, β, and κ-rings. Examination of the primary structure of lycopene β-cyclase revealed 55% identity with that of antheraxanthin κ-cyclase. Recombinant lycopene β-cyclase afforded only β-carotene, while recombinant antheraxanthin κ-cyclase catalyzed the formation of β-carotene from lycopene as well as the conversion of antheraxanthin into the κ-carotenoid capsanthin. Since the formation of β- and κ-rings involves a transient carotenoid carbocation, this suggests that both cyclases initiate and/or neutralize the incipient carbocation by similar mechanisms. Several amine derivatives protonated at physiological pH were used to examine the molecular basis of this phenomenon. The β- and κ-cyclases displayed similar inhibition patterns. Affinity or photoaffinity labeling usingp-dimethylamino-benzenediazonium fluoroborate,N,N-dimethyl-2-phenylaziridinium, and nicotine irreversibly inactivated both cyclase enzymes. Photoaffinity labeling using [3H]nicotine followed by radiosequence analysis and site-directed mutagenesis revealed the existence of two cyclase domains characterized by the presence of reactive aromatic and carboxylic amino acid residues. We propose that these residues represent the “negative point charges” involved in the coordination of the incipient carotenoid carbocations.