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International audience; Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock in plants, Drosophila and humans. Drosophila melanogaster cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD(center dot-)) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. Here, we show by kinetic modeling of the DmCry photocycle that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD(center dot-)) as the signaling state in vivo. We show by fluorescence detection techniques that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O-2) to reactive oxygen species (ROS). We extend these observations in living cells to demonstrate transient formation of superoxide (O-2(center dot-)), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role.

Neoxanthin, a precursor of the plant hormone abscisic acid, is an allenic xanthophyll recognized as the last product of carotenoid synthesis in green plants. A cDNA for neoxanthin synthase (NSY) was isolated from tomato using a molecular approach based on the mechanistic and structural similarities of NSY to two other closely related carotenogenic enzymes, lycopene cyclase (LCY) and capsanthin-capsorubin synthase (CCS). The identified tomato NSY cDNA (T.NSY) encodes a 56-kDa plastid-targeted protein that when expressed in Escherichia coli, catalyzes the conversion of violaxanthin to neoxanthin. In tobacco leaves that transiently express T.NSY, an increase in neoxanthin content with a concomitant decrease in violaxanthin is observed. NSY is structurally similar to LCY and CCS. However, in Cyanobacteria, the generally accepted progenitor of plastids, both CCS and NSY are absent while LCY is present. LCY catalyzes a simplified version of the reaction catalyzed by NSY and CCS suggesting that these two enzymes were remodeled from LCY during higher plant evolution to create new forms of oxidized carotenoids.

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B1), and pyridoxine (vitamin B6) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.

To dissect the mechanism by which carotenoid hydroxylases catalyze xanthophyll formation, we have cloned two pepper cDNAs encoding beta-cryptoxanthin and zeaxanthin biosynthetic enzymes. Using an in vitro system, we find that both enzymes are ferredoxin dependent and that their activity is strongly inhibited by iron chelators such as o-phenanthroline or 8-hydroxyquinoline. This suggests the transfer of a reducing equivalent from NADPH to the hydroxylase via ferredoxin and the involvement of an iron activated oxygen insertion process. Based on sequence analysis, the putative histidine clusters involved in the iron coordination were identified and their roles evaluated. Following site-directed mutagenesis of the identified histidine residues hydroxylase activity was totally inactivated. Collectively, our data indicate that carotenoid hydroxylases belong to a new class of diiron proteins structurally related to membrane fatty acid desaturases. Mechanistically, both types of enzymes exploit iron activated oxygen to break the C-H bond with concomitant formation of double bond or oxygen insertion. We propose that the same mechanism operates for beta-carotene ketolase and probably for other carotenoid oxygenases as well.

The addition of phytyl side chain to chlorophylls, tocopherols and phylloquinone is prerequisite to their integration into plastid membranes. We have cloned a cDNA encoding a pre-geranylgeranyl reductase from Arabidopsis thaliana. The deduced primary structure predicts a mature size with a molecular mass of 47 kDa and displays a characteristic dinucleotide binding domain. Geranylgeranyl reductase expressed in Escherichia coli sequentially catalyzes the reduction of geranylgeranyl-chlorophyll a into phytyl-chlorophyll a as well as the reduction of free geranylgeranyl diphosphate into phytyl diphosphate. Due to its multifunctionality and weak hydrophobicity, we suggest that in plastid the same geranylgeranyl reductase is recruited into the chlorophyll, the tocopherol and the phylloquinone pathways. The geranylgeranyl reductase gene is up-regulated during etioplast to chloroplast and chloroplast to chromoplast development.

Later steps of carotenoid biosynthesis catalyzed by cyclase enzymes involve the formation of alpha, beta, and kappa-rings. Examination of the primary structure of lycopene beta-cyclase revealed 55% identity with that of antheraxanthin kappa-cyclase. Recombinant lycopene beta-cyclase afforded only beta-carotene, while recombinant antheraxanthin kappa-cyclase catalyzed the formation of beta-carotene from lycopene as well as the conversion of antheraxanthin into the kappa-carotenoid capsanthin. Since the formation of beta- and kappa-rings involves a transient carotenoid carbocation, this suggests that both cyclases initiate and/or neutralize the incipient carbocation by similar mechanisms. Several amine derivatives protonated at physiological pH were used to examine the molecular basis of this phenomenon. The beta-and kappa-cyclases displayed similar inhibition patterns. Affinity or photoaffinity labeling using p-dimethylamino-benzenediazonium fluoroborate, N,N-dimethyl-2-phenylaziridinium, and nicotine irreversibly inactivated both cyclase enzymes. Photoaffinity labeling using [3H]nicotine followed by radiosequence analysis and site-directed mutagenesis revealed the existence of two cyclase domains characterized by the presence of reactive aromatic and carboxylic amino acid residues. We propose that these residues represent the "negative point charges" involved in the coordination of the incipient carotenoid carbocations.

Pepper (Capsicum annuum) β-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the corresponding enzyme overexpressed and purified from Escherichia coli, for investigation of its catalytic activity. The recombinant protein did not directly accept NADPH for epoxidation of cyclohexenyl carotenoids, nor did it operate according to a peroxygenase-based mechanism. Instead, the reducing power of NADPH was transferred to the epoxidase via reduced ferredoxin as shown by reconstitution of epoxidase activity in the presence of NADPH, ferredoxin oxidoreductase, and ferredoxin. Bacterial rubredoxin could be substituted for ferredoxin. The pepper epoxidase acted specifically on the β-ring of xanthophylls such as β-cryptoxanthin, zeaxanthin, and antheraxanthin. The proposed reaction mechanism for epoxidation involves the formation of a transient carbocation. This characteristic allows selective inhibition of the epoxidase activity by different nucleophilic diethylamine derivatives, p-dimethylaminobenzenediazonium fluoroborate and N,N-dimethyl-2-phenylaziridinium. It was also shown that the epoxidase gene was up-regulated during oxidative stress and when chloroplasts undergo differentiation into chromoplasts in pepper fruit.

Cysteine synthase (O-acetylserine sulfhydrylase) has been purified to homogeneity from bell pepper (Capsicum annuum) fruit chromoplasts. This enzyme consists of two subunits of 35 kDa. Immunocytochemical localization experiments confirmed the plastid location of this enzyme. A full-length cDNA was isolated from an expression library of C. annuum. The deduced peptide sequence revealed high similarity between the C. annuum cysteine synthase and its bacterial counterparts. In vitro transcription and translation of the cDNA and subsequent import experiments demonstrated that the encoded cysteine synthase is located in the plastids. The steady-state level of the cysteine synthase mRNA is almost constant in dark-grown hypocotyls, leaves, and fruits. However, a slight increase in this mRNA level was detected during fruit development (when the 25 S rRNA was taken as an internal standard). Similarly, the cysteine synthase activity in plastids was found to increase during fruit development and reaches the highest levels in the chromoplasts of red fruits. To address the physiological role of this phenomenon, we have shown that cysteine is engaged in the active metabolism of glutathione. Thus, in connection with the previous demonstration of an active tocopherol metabolism, it is concluded that differentiation of chloroplast to chromoplast in C. annuum involves an active synthesis of potential antioxidants or redox modulators.

The nature of isoprenoids synthesized in plants is primarily determined by the specificity of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non-secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy-xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non-green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.

Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation.

Summary The late steps of carotenoid biosynthesis in plants involve the formation of xanthophyUs. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xantho- phyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6.epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionallty, the name capsanthin-capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a full- length cDNA clone encoding a capsanthin capserubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capeanthin- capsorubin synthase gene is specifically expressed during chromoplast development in fruits accumu- lating ketocarotenoids, but not in mutants impaired in this biosynthetic step. phytofluene desaturase (Hugueney

Background: The etiology of stroke in sickle cell anemia (SCA) is complex and multiple genetic factors outside of the sickle gene undoubtedly contribute to stroke risk in SCA. Simultaneous analyses of multiple genetic variants may therefore be more informative than evaluation of individual candidate genes. Moreover, stroke in SCA is phenotypically heterogeneous, varying from large cortical vessel (LV) distribution infarcts to milder lacunar infarcts due to small vessel disease, suggesting distinct pathogenetic mechanisms for stroke in SCA. We previously reported risk associations with particular TNFA, IL4R and ADRB2 polymorphisms in children enrolled in the CSSCD. Methods: To confirm our previous findings from the CSSCD and identify other potential “susceptibility” loci that may influence the development of LV stroke, we extended our investigations to SCA children who participated in the Stroke Prevention Trial in Sickle Cell Anemia (STOP). Children with MRA evidence of obstruction or stenosis of one or more large vessels (LV disease) were compared with untransfused children with a normal MRA. Ninety-seven patients were included in this study. Of the 119 patients originally enrolled in STOP, 77 (30 MRA+, 47 MRA−) were eligible for this study and were analyzed together with a local sample of 20 MRA(+)patients. Genotyping for 110 polymorphisms among 65 candidate cardiovascular and proinflammatory genes was performed using multiplex linear array methods. Of the 110 variant sites examined, 95 were sufficiently polymorphic to permit statistical testing, using 2 x 2 exact tests. Results: When comparing MRA(+) patients with MRA(−) patients, 9 of the polymorphic loci revealed nominally significant differences (P

Rishitin accumulation as well as its biosynthesis from added [2- 14 C]MVA was observed in tomato cell suspension cultures elicited by yeast extract.

We have examined the nature of the decline of lactase (EC 3.2.1.23) activity in the maturing rat intestine. It was established in an initial study that the activity decline reflected a proportional reduction in the concentration of the enzyme protein. Accumulation patterns into lactase, total intestinal proteins and sucrase (Ec 3.2.1.48)-isomaltase (EC 3.2.1.10) were compared, 4 h following administration of a tracer dose of [ 3 H]leucine to weanling rats exhibiting a wide range of lactase decline. Accumulation of increasing amounts of label in total intestinal proteins and sucrase-isomaltase pools was found to accompany the lactase decline, in contrast to accumulation of a constant amount of label in the declining lactase pools. The pattern of increased label accumulation in total intestinal proteins was shown in a corollary study to reflect a corresponding acceleration of total protein synthesis. On this basis, the finding of a constant amount of label in the declining lactase pools suggested a constant synthesis of lactase. We proposed earlier that associated reductions in enterocyte life-span (leading to correspondingly less lactase accumulation) rather than suppressed synthesis may provide the primary causal basis of lactase decline in the postweaned mammal.

Hydrocortisone administration to infant rats enhanced cellobiase and maltase activities and induced precocious expression of sucrase and trehalase activities along the length of the small intestine. These activity changes reflected proportional concentration increases in the enzymes lactase (EC 3.2.1.23), maltase/glucoamylase (EC 3.2.1.20) and sucrase-isomaltase (EC 3.2.1.48/10). Administration of an equivalent tracer dose of [3H]leucine (by body weight) to control and hydrocortisone-treated infant rats resulted in greater accumulation of label in the carbohydrase pools of the treated rats, suggesting their increased de novo synthesis. The increased concentrations of lactase and maltase/glucoamylase induced by exogenous hydrocortisone were matched by the presence of corresponding greater amounts of label in their brush border pools. Accumulation of label in each of the lactase, maltase/glucoamylase and sucrase-isomaltase pools was generally similar in the hydrocortisone-treated rats, suggesting equivalent stimulation of their synthesis as a group by the humoral agent. The turnover rates of the carbohydrases as a group were found to be similar and did not appear to differ in control and hydrocortisone-treated rats. Total protein synthesis rates were slightly greater in the intestine of the hydrocortisone-treated group of rats.

The cyclization of lycopene to β,β-carotene (β-carotene) was studied using chromoplast membranes prepared from Capsicum fruits. The enzymic activity is tightly bound to the membranes and, under optimal conditions, lycopene is cyclized to β-carotene without accumulation of the intermediate β,ψ-carotene (γ-carotene). In this system, no formation of (6,R)-β,ϵ-carotene (α-carotene) occurs. The optimal pH for lycopene cyclization is 6.8. Nucleotide cofactors are not required for activity. The enzymic activity is stimulated in the presence of Tween 80 in contrast to the inhibitory effect of Triton X-100. Thiol reagents such as mersalyl, N-ethylmaleimide and p-chloromercuribenzoate strongly inhibit the cyclization of lycopene to β-carotene. Octyldiethylamine and dodecyl diethylamine are potent inhibitors of lycopene cyclase activity from Capsicum chromoplast membranes.

Cells of the intestinal mucosa of the infant and adult rat maintain a pattern of continuous growth, accumulating structural and functional proteins and lipids while migrating the length of the villus column. Cells of jejunal and ileal segments were fractionated sequentially from villus tip to inner crypt and distribution patterns were determined for DNA, total protein, cholesterol, phospholipid, and disaccharidases. Patterns of increasing ratios of protein, lipids, and disaccharidases to DNA were maintained to villus tips, with only slight fall-off of enzymes observed. Distribution profiles of disaccharidases, when computed relative to protein (as seen in previous reports), show distortion of the true cellular distribution pattern of these enzymes as determined by the DNA content of the fractions. Wide variation in cell protein concentrations was evident between jejunal and ileal segments in pre- and postweaned rats. Ileal cells of the suckling rat contained particularly high protein concentrations, which appeared to be largely transitory in nature and related to food intake. Cholesterol and phospholipids were found to be concentrated in the microvillus membrane and account for a significantly large fraction of the cellular content of these lipids.

gamma-Tocopherol methyltransferase was solubilized and purified from Capsicum chromoplast membranes by a combination of standard fractionation techniques. The purified enzyme was electrophoretically homogeneous, and its molecular weight, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 33,000. In the absence of detergent, the enzyme formed high molecular weight aggregates. Several properties of the enzyme have been determined. The Km values were 2.5 and 13.7 microM for S-adenosylmethionine and gamma-tocopherol, respectively. The enzyme was able to transfer the methyl group S-adenosylmethionine to N-4-azido-2-nitrophenyl-beta-alanyl-gamma-tocopherol. The rate of transfer was less efficient compared to gamma-tocopherol. In the presence of ultraviolet light, this analog inhibited the gamma-tocopherol methyltransferase activity.

Phytoene is the first C 40 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 ( ii ) 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 ( M r 47,500) were demonstrated by NaDodSO 4 /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 Michaelis-Menten kinetics, with K m values of 0.30 μM and 0.27 μM, respectively, for geranylgeranyl diphosphate and prephytoene diphosphate. The activity of the enzyme depends strictly upon the presence of Mn 2+ . 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.

Through the use of Capsicum chromoplast membranes, we report for the first time the direct methylation of γ-tocopherol into α-tocopherol in the presence of S-adenosylmethionine. Furthermore the S-adenosylmethionine: γ-tocopherol methyltransferase activity has been solubilized. On a protein basis, the activity recovered in the soluble preparation was higher than that bound to the membranes.

The synthesis of alpha-tocopherol from 2,3-dimethylphytylquinol and S-adenosyl-l-methionine was achieved using Capsicum annuum fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:gamma-tocopherol methyl-transferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction.

2-Aza-2,3-dihydrosqualene strongly inhibited lycopene cyclase from Capsicum chromoplast membranes.