Abstract
The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-α/β-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates. The reaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene β-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction.
Highlights
Carotenoids belong to a large isoprenoid family, some members of which are indispensible in all photosynthetic organisms; they can be formed by some heterotrophic bacteria and fungi
The lines of thinking developed for CRTY might apply for CRTISO: It was shown that lycopene cyclization depended on reduced FAD involved in the stabilization of a transition state
Inspired by results obtained with two other isomerases involved in isoprenoid metabolism, namely type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2; see Ref. 27 and citations therein) and lycopene -cyclase (CRTY/LCY-b; 20, 21) both catalyzing FADred-dependent non-redox reactions, we hypothesized a similar mechanism operating with CRTISO
Summary
Chemicals Used—Flavin cofactors modified at various positions have been detailed in a compiled report [22]. For the identification of protein-bound cofactors, CRTISO was purified essentially following the procedures described above, glycerol and Tween 20 were omitted from the elution buffer. The standard CRTISO assay mixture (final volume 200 l) consisted of 163 l of buffer A which was supplemented with typically 30 l of the substrate-containing liposome suspension to result in a final carotene concentration of 5 M. Because the reaction conditions of OsLCY-b are essentially identical with those of CRTY including anaerobosis [20], 5 g of IMAC-purified OsLCY-b protein was added to the standard CRTISO assays. Refer to Gradinaru et al [26]
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