Plant carotene cis-trans isomerase CRTISO : a new member of the FAD_RED-dependent flavoproteins catalyzing non-redox reactions

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 desaturaseCRTI, 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 occupiedby FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtainedwith C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphateisomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolismbelonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kineticsof 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 introducedinto 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.