Carotenoid biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing the synthesis of phytoene

Abstract

Phytoene is the frst C40 intermediate in the biogenesis of carotenoids. It is formed by two enzyme activities, catalyzing (i) the coupling of two molecules of geranylgeranyl diphosphate to yield prephytoene diphosphate and (i) the conversion of prephytoene diphosphate into phytoene. We show now, with Capsicum chromoplast stroma, that the overall activity resides in a single protein, which has been purified to homogeneity by affinity chromatography. The monomeric structure and the molecular size (Mr 47,500) were demonstrated by NaDodSO4/PAGE and glycerol gradient centrifugation. Further characterization was achieved by using specific antibodies which allowed immunofractionation and immunoprecipitation of the enzymatic activity from chromoplast stroma. The two reactions followed conventional MichaelisMenten kinetics, with Km values of 0.30 ,.M and 0.27 IAM, respectively, for geranylgeranyl diphosphate and prephytoene diphosphate. The activity of the enzyme depends strictly upon the presence ofMn2. This selectivity may be one of the factors regulating the competition with potentially rival enzymes converting geranylgeranyl diphosphate into other plastid terpenoids. The two enzymatic reactions were inhibited by inorganic pyrophosphate and by the arginine-specific reagent hydroxyphenylglyoxal. In no instance were the two reactions kinetically uncoupled. These properties strongly suggest that the same enzyme catalyzes the two consecutive reactions, and we propose to name it phytoene synthase.Carotenoids are required for the normal differentiation and function of plastids. Phytoene, the first C40 intermediate in the carotenoid pathway, is synthesized by a soluble enzymatic complex localized in plastid stroma (1)(2)(3)(4)(5), by the multistep process outlined in Scheme I. The early steps, for which the individual enzymes were recently isolated and purified (6), are the isomerization of isopentenyl diphosphate (1) into dimethylallyl diphosphate (2), followed by three prenylations forming successively geranyl diphosphate (3), farnesyl diphosphate (4), and geranylgeranyl diphosphate (5). This sequence of reactions is common to all terpenoids, including other plastid terpenoids (chlorophylls, plastoquinones, tocopherols, phylloquinones, and polyterpenes). On the other hand, the next two steps, in which prephytoene diphosphate (6) and phytoene (7) are formed, are catalyzed by the first carotenoid-specific enzymes in the pathway, tentatively termed "geranylgeranyl pyrophosphate geranylgeranyltransferase" and "phytoene synthase" (7) since they have been neither isolated nor characterized previously from any other source (5,8,9). Therefore, the identification of the enzyme components catalyzing these two steps is crucial for our understanding of the molecular mechanism of carotenoid biosynthesis and of its regulation; a particularly interesting The present paper answers this question. We show, with Capsicum chromoplasts, previously selected for studies of carotenogenesis (3,(10)(11)(12), that a single monomeric protein ...

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6‐epoxycarotenoid into ketocarotenoid

“…On the basis of this finding, we propose the name capsanthin-capsorubin synthase (CCS) for this ketoxanthophyll synthase which, like other carotenogenic enzymes already characterized from C. annuum, i.e. GGPPS , phytoene synthase (Dogbo et aL, 1988) and phytoene Stromal and membrane protein fractions (50 pg) from isolated chloroplasts and chromoplasts were fractionated using a 10% (w/v) SDSpolyacrylamide gel stained with Coomassie bdlliant blue, except for purified CCS which was stained by silver nitrate. Lane 1, chloroplast stroma; lane 2, chromoplast stroma; lane 3, chloroplast membranes; lane 4, total chromoplast membranes; lane 5, purified membranes; lane 6, purified CCS.…”

“…This system has allowed the characterization and cloning of geranylgeranyl pyrophosphate synthase (GGPPS) Kuntz et aL, 1992), phytoene synthase (Dogbo et aL, 1988;R6mer et aL, 1993) and phytoene- (a) Internal chmmoplast structures observed in situ after silver-proteinate poststaining (bar = 0.34 IJm). Spurr and Harris, 1968).…”

Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase.

“…The similarity between squalene synthase and phytoene synthase have been pointed out (15,17,30). Since the latter enzyme prefers Mn2+ as a cofactor rather than Mg2+, the similarity of the two enzymes is maximum in this secondary reaction.…”

“…This enzyme is similar to squalene synthase in that it is bifunctional, converting two molecules of geranylgeranyl diphosphate to phytoene through prephytoene diphosphate, but different in that it is located in plastids and never utilizes NADPH (30). The amino acid sequences are only 20% identical (over 165 amino acids in the middle region) in the Arabidopsis enzyme and tomato phytoene synthase (31).…”

“…Since the latter enzyme prefers Mn2+ as a cofactor rather than Mg2+, the similarity of the two enzymes is maximum in this secondary reaction. In contrast, phytoene synthase cannot generate a squalene homolog (dihydrophytoene), even in the presence of NADPH and Mg2+ (30). Conclusion and Perspectives.…”