[Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum].

Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced ß-sandwich fold. A large ß-sheet( ß4-ß11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning ß4,ß5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

An In Vitro Enzyme System for the Production of myo-Inositol from Starch.

We developed an in vitro enzyme system to produce myo-inositol from starch. Four enzymes were used, maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase (MIPS), and inositol monophosphatase (IMPase). The enzymes were thermostable: MalP and PGM from the hyperthermophilic archaeon Thermococcus kodakarensis, MIPS from the hyperthermophilic archaeon Archaeoglobus fulgidus, and IMPase from the hyperthermophilic bacterium Thermotoga maritima The enzymes were individually produced in Escherichia coli and partially purified by subjecting cell extracts to heat treatment and removing denatured proteins. The four enzyme samples were incubated at 90°C with amylose, phosphate, and NAD+, resulting in the production of myo-inositol with a yield of over 90% at 2 h. The effects of varying the concentrations of reaction components were examined. When the system volume was increased and NAD+ was added every 2 h, we observed the production of 2.9 g myo-inositol from 2.9 g amylose after 7 h, achieving gram-scale production with a molar conversion of approximately 96%. We further integrated the pullulanase from T. maritima into the system and observed myo-inositol production from soluble starch and raw potato with yields of 73% and 57 to 61%, respectively.IMPORTANCEmyo-Inositol is an important nutrient for human health and provides a wide variety of benefits as a dietary supplement. This study demonstrates an alternative method to produce myo-inositol from starch with an in vitro enzyme system using thermostable maltodextrin phosphorylase (MalP), phosphoglucomutase (PGM), myo-inositol-3-phosphate synthase, and myo-inositol monophosphatase. By utilizing MalP and PGM to generate glucose 6-phosphate, we can avoid the addition of phosphate donors such as ATP, the use of which would not be practical for scaled-up production of myo-inositol. myo-Inositol was produced from amylose on the gram scale with yields exceeding 90%. Conversion rates were also high, producing over 2 g of myo-inositol within 4 h in a 200-ml reaction mixture. By adding a thermostable pullulanase, we produced myo-inositol from raw potato with yields of 57 to 61% (wt/wt). The system developed here should provide an attractive alternative to conventional methods that rely on extraction or microbial production of myo-inositol.

The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles.

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1(-) mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

Molecular cloning and transgenic characterization of the genes encoding chalcone synthase and chalcone isomerase from the Tibetan herbal plant Mirabilis himalaica.

Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase (MhCHS) and chalcone isomerase (MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures.

Lycopene cyclase and phytoene synthase activities in the marine yeast Rhodosporidium diobovatum are encoded by a single gene crtYB.

crtYB, encoding lycopene cyclase and phytoene synthase was cloned from Rhodosporidium diobovatum ATCC 2527 by rapid amplification of cDNA ends method. The full-length cDNA of crtYB is 2, 330 bp and contains eight introns. The gene products is a 594 amino acids, with a predicted molecular mass of 65.63 kDa and a pI of 6.73. The N-terminus of the protein contains six transmembrane regions, which has been characterized as a lycopene beta-cyclase. The C-terminal half has squalene and phytoene synthase signatures that identified as phytoene synthetase. By heterologous complementary detection of this gene in E. coli and HPLC analysis, the regions responsible for phytoene synthesis and lycopene cyclization were localized within the protein.

cDNA cloning of a novel gene codifying for the enzyme lycopene ß-cyclase from Ficus carica and its expression in Escherichia coli.

Lycopene beta-cyclase (ß-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic ß-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, ß-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-ß from Ficus carica (Lyc-ß Fc), which codes for the enzyme lycopene ß-cyclase (ß-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. ß-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to ß-carotene as a result of the enzyme's action. The ß-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the ß-carotene. The lycopene to ß-carotene conversion rate was 90%. The experiments carried out in this work showed that ß-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to ß-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing ß-LCY in E. coli, we have obtained a new gene for ß-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica.

Characterization of cDNA of lycopene beta-cyclase responsible for a high level of beta-carotene accumulation in Dunaliella salina.

Lycopene beta-cyclase (Lyc-B) is the key enzyme in the catalysis of linear lycopene to form cyclic beta-carotene, an indispensable part of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Studies showing that the microalga Dunaliella salina can accumulate a high level of beta-carotene are lacking. We hypothesize that D. salina is closely involved with the catalytic mechanism of Lyc-B and the molecular regulation of its gene. In this study, we used RT-PCR and RACE-PCR to isolate a 2475 bp cDNA with a 1824 bp open reading frame, encoding a putative Lyc-B, from D. salina. Homology studies showed that the deduced amino acid sequence had a significant overall similarity with sequences of other green algae and higher plants, and that it shared the highest sequence identity, up to 64%, with Lyc-B of Chlamydomonas reinhardtii. Codon analysis showed that synonymous codon usage in the enzyme has a strong bias towards codons ending with adenosine. Two motifs were found in the Lyc-B sequence, one at the N terminus, for binding the hypothetical cofactor FAD, and the other was a substrate carrier motif in oxygenic organisms shared by an earlier carotenogenesis enzyme, phytoene desaturase, and Lyc-B. A tertiary structure prediction suggested that the catalytic or binding site structure within LycB from D. salina is superior to that of both H. pluvialis and C. reinhardtii. The LycB protein from D. salina was quite removed from that of H. pluvialis and C. reinhardtii in the phylogenetic tree. Taken as a whole, this information provides insight into the regulatatory mechanism of Lyc-B at the molecular level and the high level of beta-carotene accumulation in the microalga D. salina.

Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast.

We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1). The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 degrees C and exhibited K(m) values of 0.57 mm and 8 microm for glucose 6-phosphate and NAD(+), respectively. NH(4)(+) and K(+) were better activators than other cations tested (Na(+), Li(+), Mg(2+), Mn(2+)), and Zn(2+) strongly inhibited activity. Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase (ino1Delta/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.

Monoterpene biosynthesis in lemon (Citrus limon). cDNA isolation and functional analysis of four monoterpene synthases.

Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)-limonene (two cDNAs) (-)-beta-pinene and gamma-terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn(2+) as bivalent metal ion cofactor resulted in higher activity than Mg(2+), with an optimum concentration of 0.6 mm. K(m) values ranged from 0.7 to 3.1 microm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.

Involvement of NADPH in the cyclization reaction of carotenoid biosynthesis.

Cyclic carotenoids, e.g. beta-carotene, are formed by cyclization of an acyclic precursor, lycopene. The gene, crtY, which encodes lycopene beta-cyclase, has a partial sequence characteristic of a pyridine nucleotide binding domain, and NAD(P)H has been reported to be an absolute requirement for the cyclization reaction in vitro. By complementary incubations with lycopene as substrate and with (4R)-[4-(2)H]NADPH in (1)H(2)O or with unlabelled NADPH in (2)H(2)O in the presence of the purified enzyme, it has now been shown that the hydrogen atom introduced at C(2) in the cyclization comes from water and not from NADPH. The previously proposed mechanism involving the initiation of cyclization by H(+) attack at C(2) of the folded acyclic end group of the precursor is thus confirmed. No hydrogen is transferred from NADPH, which is therefore not involved directly in the cyclization reaction, but must play an indirect role, e.g. as an allosteric activator.

One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases.

Carotenoids in the photosynthetic membranes of plants typically contain two beta-rings (e.g., beta-carotene and zeaxanthin) or one epsilon- and one beta-ring (e.g., lutein). Carotenoids with two epsilon-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene epsilon-cyclase (LCYe) adds one epsilon-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene beta-cyclase (LCYb) adds two beta-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis epsilon-cyclases involved in the determination of ring number were mapped by analysis of chimeric epsilon-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one epsilon-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon-rings. An R residue in this position also yields a bi-epsilon-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.