Characterization of Plant Carotenoid Cyclases as Members of the Flavoprotein Family Functioning with No Net Redox Change    

Abstract

The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene b-cyclase (LCY-B), which converts lycopene into b-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of k-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to b-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of b-carotene and k-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of b-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

“…This avoids contamination of the system as observed when tributylamine is used as counter ion68. Furthermore, the FAD and FMN peaks were without tailing in contrast to what has been observed with C18 chromatography69.…”

Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

“…Although the similarity of the cyclases from non-photosynthetic bacteria (crtY) with those from cyanobacteria (CrtL) and plants (LCYB and LCYE) is rather low, recent results indicate that, similar to CRTISO, both types of carotenoid cyclases are flavoenzymes that require the reduced form of the FAD cofactor despite catalyzing their reactions with no net redox (Mialoundama et al, 2010;Yu et al, 2010). The existence of some conserved sequence patterns further suggests a common phylogenetic origin (Krubasik and Sandmann, 2000).…”

“…Because the carotenoid desaturases PDS and ZDS use plastoquinone as hydrogen acceptor, their activity is connected to the photosynthetic electron transport chain (Carol and Kuntz, 2001). Both PDS and ZDS, as well as other carotenoid biosynthetic enzymes such as CRTISO, LCYB, LCYE, and ZEP, contain a conserved FADbinding motif that suggests the involvement of redox balance in the corresponding enzymatic reactions (Hugueney et al, 1992;Büch et al, 1995;Marin et al, 1996;Schnurr et al, 1996;Isaacson et al, 2004;Mialoundama et al, 2010;Yu et al, 2010;Yu et al, 2011). There is also evidence for a redox control of the expression of some carotenoid biosynthetic genes in tobacco chloroplasts and tomato fruit chromoplasts (Woitsch and Römer, 2003;Nashilevitz et al, 2010).…”

Plant Carotene Cis-Trans Isomerase CRTISO

“…The 5-cis configuration of lycopene may forbid cyclization because this C(5)-C(6) double bond is mechanistically involved in lycopene cyclization (20,21) while double bonds in a more central position are not. 9-cis-configured double bonds are especially interesting since 9-cis configured xanthophylls represent a molecular "earmark" to direct them into the biosynthesis of abscisic acid (ABA) through the action of 9-mono-cis epoxycarotenoid oxygenases (NCEDs).…”

“…Therefore, the lines of thinking developed for CRTY might also apply for CRTISO: It was shown that lycopene cyclization depended on reduced FAD involved in the stabilization of a transition state. A specific glutamate was also needed that is believed to be involved in acid/base catalysis (20,21). Based on this, it was suggested that the sole function of respiratory chain activities in vitro was to ensure reducing conditions (anaerobiosis), i.e.…”