MOLECULAR ANALYSIS OF CAROTENOID CYCLASE INHIBITION

Later steps of carotenoid biosynthesis catalyzed by cyclase enzymes in volve the formation of alpha, beta, and kappa-rings, Examination of th e primary structure of lycopene beta-cyclase revealed 55% identity wit h that of antheraxanthin kappa-cyclase. Recombinant lycopene beta-cycl ase afforded only beta-carotene, while recombinant antheraxanthin kapp a-cyclase catalyzed the formation of beta-carotene from lycopene as we ll as the conversion of antheraxanthin into the kappa-carotenoid capsa nthin. Since the formation of beta- and kappa-rings involves a transie nt carotenoid carbocation, this suggests that both cyclases initiate a nd/or neutralize the incipient carbocation by similar mechanisms, Seve ral amine derivatives protonated at physiological pH were used to exam ine the molecular basis of this phenomenon. The beta- and kappa-cyclas es displayed similar inhibition patterns. Affinity or photoaffinity la beling using p-dimethylamino-benzenediazonium fluoroborate, N,N-dimeth yl-2-phenylaziridinium, and nicotine irreversibly inactivated both cyc lase enzymes, Photoaffinity labeling using [H-3]nicotine followed by r adiosequence analysis and site-directed mutagenesis revealed the exist ence of two cyclase domains characterized by the presence of reactive aromatic and carboxylic amino acid residues. We propose that these res idues represent the ''negative point charges'' involved in the coordin ation of the incipient carotenoid carbocations. (C) 1997 Academic Pres s.