Musashi2 promotes the progression of pancreatic cancer through a novel ISYNA1-p21/ZEB-1 pathway.

Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol-3-phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with MSI2 expression, T stage, vascular permeation and poor prognosis in PC patients. What's more, patients expressed high MSI2 and low ISYNA1 level had a significant worse prognosis. And in wtp53 Capan-2 and SW1990 cells, ISYNA1 was downregulated by p53 silencing. ISYNA1 silencing promoted cell proliferation and cell cycle by inhibiting p21 and enhanced cell migration and invasion by upregulating ZEB-1. However, MSI2 silencing upregulated ISYNA1 and p21 but downregulated ZEB-1, which can be rescued by ISYNA1 silencing. Moreover, reduction of cell migration and invasion resulting from MSI2 silencing was significantly reversed by ISYNA1 silencing. In summary, MSI2 facilitates the development of PC through a novel ISYNA1-p21/ZEB-1 pathway, which provides new gene target therapy for PC.

Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.

Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues.

The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.

Kemp Eliminase Activity of Ketosteroid Isomerase.

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

Scale translation from shaken to diffused bubble aerated systems for lycopene production by Blakeslea trispora under stimulated conditions.

This study deals with the scale up of Blakeslea trispora culture from the successful surface-aerated shake flasks to dispersed-bubble aerated column reactor for lycopene production in the presence of lycopene cyclase inhibitor 2-methyl imidazole. Controlling the initial volumetric oxygen mass transfer coefficient (kLa) via airflow rate contributes to increasing cell mass and lycopene accumulation. Inhibitor effectiveness seems to decrease in conditions of high cell mass. Optimization of crude soybean oil (CSO), airflow rate, and 2-methyl imidazole was arranged according to central composite statistical design. The optimized levels of factors were 110.5 g/L, 2.3 vvm, and 29.5 mg/L, respectively. At this optimum setting, maximum lycopene yield (256 mg/L) was comparable or even higher to those reported in shake flasks and stirred tank reactor. 2-Methyl imidazole use at levels significantly lower than those reported for other inhibitors in the literature was successful in terms of process selectivity. CSO provides economic benefits to the process through its ability to stimulate lycopene synthesis, as an inexpensive carbon source and oxygen vector at the same time.

Perturbation of the Vacuolar ATPase: A NOVEL CONSEQUENCE OF INOSITOL DEPLETION.

Depletion of inositol has profound effects on cell function and has been implicated in the therapeutic effects of drugs used to treat epilepsy and bipolar disorder. We have previously shown that the anticonvulsant drug valproate (VPA) depletes inositol by inhibiting myo-inositol-3-phosphate synthase, the enzyme that catalyzes the first and rate-limiting step of inositol biosynthesis. To elucidate the cellular consequences of inositol depletion, we screened the yeast deletion collection for VPA-sensitive mutants and identified mutants in vacuolar sorting and the vacuolar ATPase (V-ATPase). Inositol depletion caused by starvation of ino1Δ cells perturbed the vacuolar structure and decreased V-ATPase activity and proton pumping in isolated vacuolar vesicles. VPA compromised the dynamics of phosphatidylinositol 3,5-bisphosphate (PI3,5P2) and greatly reduced V-ATPase proton transport in inositol-deprived wild-type cells. Osmotic stress, known to increase PI3,5P2 levels, did not restore PI3,5P2 homeostasis nor did it induce vacuolar fragmentation in VPA-treated cells, suggesting that perturbation of the V-ATPase is a consequence of altered PI3,5P2 homeostasis under inositol-limiting conditions. This study is the first to demonstrate that inositol depletion caused by starvation of an inositol synthesis mutant or by the inositol-depleting drug VPA leads to perturbation of the V-ATPase.

Ethylbutyrate, a valproate-like compound, exhibits inositol-depleting effects--a potential mood-stabilizing drug.

AIMS: To screen for inositol-depleting valproate-like compounds as potential mood stabilizing drugs. MAIN METHODS: We exploited the yeast Saccharomyces cerevisiae, as a model in which inositol de novo synthesis has been extensively characterized, to test the effects of ethyl butyrate (EB), 2-ethyl-butyric acid, sodium butyrate, and n-propyl hexanoate on inositol biosynthesis. Cell growth was followed by measuring the optical density of the cultures (spectrophotometrically), RNA abundance was determined by Northern blot analysis, intracellular inositol was measured by a fluorometric assay, and 1-d-myo-inositol-3-phosphate synthase activity was examined using a chromatographic method. KEY FINDINGS: Of the tested compounds, only EB exhibited an inositol-depleting effect. The inositol-depleting effect of EB was achieved without significant adverse effect on cell growth, pointing to lesser toxicity compared to valproate. SIGNIFICANCE: These results indicate that EB is a potential candidate for mood-stabilizing therapy.

Lycopene production using Blakeslea trispora in the presence of 2-methyl imidazole: yield, selectivity, and safety aspects.

The potential role of 2-methyl imidazole in improving lycopene production by Blakeslea trispora with regards to yield, selectivity, and safety aspects was investigated in batch culture. Optimization of the bioprocess conditions in terms of (a) (+) and (-) strain ratio in the inoculum, (b) initial crude soybean oil (CSO) addition level, and (c) the amount of 2-methyl imidazole was based on response surface methodology to achieve maximum lycopene production. The dependence of growth kinetics, lycopene yield, and selectivity of the bioprocess on the above factors was clear. 2-Methyl imidazole at 50 mg/L was found equally active in terms of lycopene cyclase inhibition with that at 200 or 100 mg/L; in all cases, lycopene accounted for 94% of the total carotenoids. The highest yield was observed at a 50 mg/L level of addition (24 mg/g of biomass dry weight,) in a substrate supplemented with CSO (48 g/L of culture medium) and inoculated with 1(+)/7(-) strain ratio.

Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions.

Haematococcus pluvialis, a green alga, accumulates carotenoids, predominantly astaxanthin, when exposed to stress conditions. In the present work, changes in the pigment profile and expression of carotenogenic genes under various nutrient stress conditions and their regulation were studied. Nutrient stress and higher light intensity in combination with NaCl/sodium acetate (SA) enhanced total carotenoid and total astaxanthin content to 32.0 and 24.5 mg g(-1) of dry biomass, respectively. Expression of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), beta-carotene ketolase (BKT), and beta-carotene hydroxylase (CHY) were up-regulated under all the stress conditions studied. However, the extent of expression of carotenogenic genes varied with stress conditions. Nutrient stress and high light intensity induced expression of astaxanthin biosynthetic genes, BKT and CHY, transiently. Enhanced expression of these genes was observed with SA and NaCl/SA, while expression was delayed with NaCl. The maximum content of astaxanthin recorded in cells grown in medium with SA and NaCl/SA correlated with the expression profile of the astaxanthin biosynthetic genes. Studies using various inhibitors indicated that general carotenogenesis and secondary carotenoid induction were regulated at both the transcriptional and the cytoplasmic translational levels. The induction of general carotenoid synthesis genes was independent of cytoplasmic protein synthesis while BKT gene expression was dependent on de novo protein synthesis.

Enhancing the carotenoid content of Brassica napus seeds by downregulating lycopene epsilon cyclase.

The accumulation of carotenoids in higher plants is regulated by the environment, tissue type and developmental stage. In Brassica napus leaves, beta-carotene and lutein were the main carotenoids present while petals primarily accumulated lutein and violaxanthin. Carotenoid accumulation in seeds was developmentally regulated with the highest levels detected at 35-40 days post anthesis. The carotenoid biosynthesis pathway branches after the formation of lycopene. One branch forms carotenoids with two beta rings such as beta-carotene, zeaxanthin and violaxanthin, while the other introduces both beta- and epsilon-rings in lycopene to form alpha-carotene and lutein. By reducing the expression of lycopene epsilon-cyclase (epsilon-CYC) using RNAi, we investigated altering carotenoid accumulation in seeds of B. napus. Transgenic seeds expressing this construct had increased levels of beta-carotene, zeaxanthin, violaxanthin and, unexpectedly, lutein. The higher total carotenoid content resulting from reduction of epsilon-CYC expression in seeds suggests that this gene is a rate-limiting step in the carotenoid biosynthesis pathway. epsilon-CYC activity and carotenoid production may also be related to fatty acid biosynthesis in seeds as transgenic seeds showed an overall decrease in total fatty acid content and minor changes in the proportions of various fatty acids.

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate.

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (+/-)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

Preferential inhibition of the lycopene epsilon-cyclase by the substituted triethylamine compound MPTA in higher plants.

In addition to the usual complement of carotenoids found in the plant leaf tissues, lettuce (Lactuca sativa), unusually, possesses large amounts of the diol lactucaxanthin. This carotenoid possesses two epsilon-end-groups and its presence provides a good model in which to study the effects of the substituted triethylamine compound 2-(4-methylphenoxy)triethylamine (MPTA) on the cyclisation of beta- and epsilon-end-groups during the biosynthesis of carotenoids. Treatment with 10 or 20microM MPTA significantly reduced levels of both beta-carotene and neoxanthin (up to 18-fold), whilst levels of violaxanthin and lutein were less affected (4-fold reduction). In contrast, levels of lactucaxanthin were not reduced even at the highest inhibitor concentration, and at 10microM MPTA levels of this xanthophyll doubled. The pigment stoichiometry of the bulk light-harvesting complex (LHCIIb) isolated from treated plants shows that lactucaxanthin successfully substituted for lutein and neoxanthin in two of the xanthophyll binding sites, namely L2 and N1. Inhibition of cyclisation was accompanied by the accumulation of lycopene and trace amounts of delta-carotene and a number of oxygenated derivatives of these precursors. Two forms of mono-hydroxy lycopene were identified together with mono-epoxy delta-carotene.

Identification and characterization of the pseudopterosin diterpene cyclase, elisabethatriene synthase, from the marine gorgonian, Pseudopterogorgia elisabethae.

The pseudopterosins are diterpene glycosides isolated from the marine gorgonian, Pseudopterogorgia elisabethae, which exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. Previously, we isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. Identification of this metabolite, and the conversion of labeled geranylgeranyl diphosphate to elisabethatriene, provided us with an assay to guide the isolation of the enzyme responsible for this cyclization. The soluble protein preparation from P. elisabethae has been partially purified (approximately 15,000-fold) using a combination of low-resolution anion-exchange, low-resolution hydrophobic interaction, high-resolution hydroxyapatite, and high-resolution anion-exchange chromatography. The diterpene cyclase was identified by comparing the molecular weight from gel permeation chromatography (approximately 47,000Da) with those of protein bands from purified fractions using SDS-PAGE gel electrophoresis. Kinetic analysis and evaluation of amino acid inhibition studies indicated that the enzyme displays similar characteristics to other terpenoid cyclases isolated from terrestrial sources. This report represents the first purification and characterization of a terpene biosynthetic enzyme from a marine invertebrate.

Not all pseudouridine synthases are potently inhibited by RNA containing 5-fluorouridine.

RNA containing 5-fluorouridine has been assumed to inhibit strongly or irreversibly the pseudouridine synthases that act on the RNA. RNA transcripts containing 5-fluorouridine in place of uridine have, therefore, been added to reconstituted systems in order to investigate the importance of particular pseudouridine residues in a given RNA by inactivating the pseudouridine synthase responsible for their generation. In sharp contradiction to the assumption of universal inhibition of pseudouridine synthases by RNA containing 5-fluorouridine, the Escherichia coli pseudouridine synthase TruB, which has physiologically critical eukaryotic homologs, is not inhibited by such RNA. Instead, the RNA containing 5-fluorouridine was handled as a substrate by TruB. The E. coli pseudouridine synthase RluA, on the other hand, forms a covalent complex and is inhibited stoichiometrically by RNA containing 5-fluorouridine. We offer a hypothesis for this disparate behavior and urge caution in interpreting results from reconstitution experiments in which RNA containing 5-fluorouridine is assumed to inhibit a pseudouridine synthase, as normal function may result from a failure to inactivate the targeted enzyme rather than from the absence of nonessential pseudouridine residues.

Asymmetrically acting lycopene beta-cyclases (CrtLm) from non-photosynthetic bacteria.

Carotenoids have important functions in photosynthesis, nutrition, and protection against oxidative damage. Some natural carotenoids are asymmetrical molecules that are difficult to produce chemically. Biological production of carotenoids using specific enzymes is a potential alternative to extraction from natural sources. Here we report the isolation of lycopene beta-cyclases that selectively cyclize only one end of lycopene or neurosporene. The crtLm genes encoding the asymmetrically acting lycopene beta-cyclases were isolated from non-photosynthetic bacteria that produced monocyclic carotenoids. Co-expression of these crtLm genes with the crtEIB genes from Pantoea stewartii (responsible for lycopene synthesis) resulted in the production of monocyclic gamma-carotene in Escherichia coli. The asymmetric cyclization activity of CrtLm could be inhibited by the lycopene beta-cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA). Phylogenetic analysis suggested that bacterial CrtL-type lycopene beta-cyclases might represent an evolutionary link between the common bacterial CrtY-type of lycopene beta-cyclases and plant lycopene beta- and epsilon-cyclases. These lycopene beta-cyclases may be used for efficient production of high-value asymmetrically cyclized carotenoids.

Stomatal constraints may affect emission of oxygenated monoterpenoids from the foliage of Pinus pinea.

Dependence of monoterpenoid emission and fractional composition on stomatal conductance (G(V)) was studied in Mediterranean conifer Pinus pinea, which primarily emits limonene and trans-beta-ocimene but also large fractions of oxygenated monoterpenoids linalool and 1,8-cineole. Strong decreases in G(V) attributable to diurnal water stress were accompanied by a significant reduction in total monoterpenoid emission rate in midday. However, various monoterpenoids responded differently to the reduction in G(V), with the emission rates of limonene and trans-beta-ocimene being unaffected but those of linalool and 1,8-cineole closely following diurnal variability in G(V). A dynamic emission model indicated that stomatal sensitivity of emissions was associated with monoterpenoid Henry's law constant (H, gas/liquid phase partition coefficient). Monoterpenoids with a large H such as trans-beta-ocimene sustain higher intercellular partial pressure for a certain liquid phase concentration, and stomatal closure is balanced by a nearly immediate increase in monoterpene diffusion gradient from intercellular air-space to ambient air. The partial pressure rises also in compounds with a low H, but more than 1,000-fold higher liquid phase concentrations of linalool and 1,8-cineole are necessary to increase intercellular partial pressure high enough to balance stomatal closure. The system response is accordingly slower, and the emission rates may be transiently suppressed by low G(V). Simulations further suggested that linalool and 1,8-cineole synthesis rates also decreased with decreasing G(V), possibly as the result of selective inhibition of various monoterpene synthases by stomata. We conclude that physicochemical characteristics of volatiles not only affect total emission but also alter the fractional composition of emitted monoterpenoids.

Mode of action of new diethylamines in lycopene cyclase inhibition and in photosystem II turnover.

The new bleaching herbicidal compound N,N-diethyl-N-(2-undecynyl)amine (NDUA) is identified here as an inhibitor of lycopene cyclase and is compared with the known cyclase inhibitors N,N-diethyl-N-[2-(4-chlorophenylthio)ethyl]amine (CPTA) and N,N-diethyl-N-[2-(4-methylphenoxy)ethyl]amine (MTPA). HPLC separation of chloroplast pigments shows lycopene accumulation in NDUA treated tissue. Variation in chain length of the undecynylamine moeity of NDUA from 7 to 21 C atoms reveals an optimum of 11 to 14 C atoms for herbicidal activity. A series of seven further analogues of NDUA and CPTA reveals the structural elements necessary for inhibition of lycopene cyclase. The effect of NDUA derivatives on photosynthesis has been studied in Chlamydomonas reinhardtii. Photosynthesis is highly sensitive, particularly towards the C14 and longer chain length analogues at nanomolar concentrations. It is shown that the breakdown of photosynthesis by NDUA is due to interference with the turnover of the D1 protein of the photosystem II reaction centre that requires the continuous biosynthesis of the two reaction-centre beta-carotene moieties in the reassembly phase. The D1 protein disappearance is most marked under strong light conditions. The depletion of photosystem II occurs before total pigment bleaching. This newly recognized mechanism in herbicidal activity is also the basis for the mode of action of other lycopene cyclase inhibitors as well as phytoene desaturase inhibitors.

Regulation of carotenoid biosynthesis during tomato fruit development: expression of the gene for lycopene epsilon-cyclase is down-regulated during ripening and is elevated in the mutant Delta.

The red colour of tomato (Lycopersicon esculentum) fruits is provided by the carotenoid pigment lycopene whose concentration increases dramatically during the ripening process. A single dominant gene, Del, in the tomato mutant Delta changes the fruit colour to orange as a result of accumulation of delta-carotene at the expense of lycopene. The cDNA for lycopene epsilon-cyclase (CrtL-e), which converts lycopene to delta-carotene, was cloned from tomato. The primary structure of CRTL-E is 71% identical to the homologous polypeptide from Arabidopsis and 36% identical to the tomato lycopene beta-cyclase, CRTL-B. The CrtL-e gene was mapped to a single locus on chromosome 12 of the tomato linkage map. This locus co-segregated with the Del gene. In the wild-type tomato, the transcript level of CrtL-e decreases at the 'breaker' stage of ripening to a non-detectable level in the ripe fruit. In contrast, it increases approximately 30-fold during fruit ripening in the Delta plants. The Delta mutation does not affect carotenoid composition nor the mRNA level of CrtL-e in leaves and flowers. These results strongly suggest that the mutation Del is an allele of the gene for epsilon-cyclase. Together with previous data, our results indicate that the primary mechanism that controls lycopene accumulation in tomato fruits is based on the differential regulation of expression of carotenoid biosynthesis genes. During fruit development, the mRNA levels for the lycopene-producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase, while the mRNA levels of the genes for the lycopene beta- and epsilon-cyclases, which convert lycopene to either beta- or delta-carotene, respectively, decline and completely disappear.

Molecular analysis of carotenoid cyclase inhibition.

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.