Molecular analysis of carotenoid cyclase inhibition

Later steps of carotenoid biosynthesis catalyzed by cyclase enzymes involve the formation of alpha, beta, and kappa-rings, Examination of the primary structure of lycopene beta-cyclase revealed 55% identity with that of antheraxanthin kappa-cyclase. Recombinant lycopene beta-cyclase afforded only beta-carotene, while recombinant antheraxanthin kappa-cyclase catalyzed the formation of beta-carotene from lycopene as well as the conversion of antheraxanthin into the kappa-carotenoid capsanthin. Since the formation of beta- and kappa-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 beta- and kappa-cyclases displayed similar inhibition patterns. Affinity or photoaffinity labeling using p-dimethylamino-benzenediazonium fluoroborate, N,N-dimethyl-2-phenylaziridinium, and nicotine irreversibly inactivated both cyclase enzymes, Photoaffinity labeling using [H-3]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. (C) 1997 Academic Press.