Phenylcoumaran Benzylic Ether Reductase Research Articles

The phenylcoumaran benzylic ether reductase gene PtPCBER improves the salt tolerance of transgenic poplar through lignan-mediated reactive oxygen species scavenging

Phenylcoumaran benzylic ether reductase (PCBER) in the phenylpropane metabolic pathway plays a crucial role in controlling plant growth and development. However, its biological function in response to abiotic stress in perennial trees is still largely obscure. In this work, a phenylcoumaran benzylic ether reductase gene, PtPCBER , isolated from poplar was overexpressed in poplar. The growth and salt resistance of transgenic plants were investigated, and the possible regulatory mechanism of PtPCBER in response to abiotic stresses was verified. PtPCBER was constitutively expressed in various tissues and organs with a predominant expression in xylems. Overexpression of PtPCBER augmented the salt and oxidative stress tolerance of transgenic poplar plants. The increased salt tolerance was associated with a relatively lower chlorphyll loss and Na + accumulation, and a higher antioxidant enzyme activity and stress gene expression, in the leaves of transgenic plants. Further metabolomic analyses revealed that overexpression of PtPCBER cut down the accumulation of coniferyl alcohols and lignans which function in the reactive oxygen species (ROS) scavenging pathway to alleviate the damage caused by oxidative stress in the leaves of transgenic plants. Taken together, our results suggest that PtPCBER has an important function in plant response to salt stress, and it could be used as an ideal candidate gene for the genetic engineering of woody plants with enhance resistance to multiple abiotic stresses through improving the scavenging capacity of ROS. • Overexpression of PtPCBER promoted salt tolerance in transgenic poplar. • The salt tolerance of transgenic plants was associated with ion homeostasis changes. • The reactive oxygen species scavenging capacity was increased in transgenic poplar.

A short-chain dehydrogenase/reductase (SDR) detection for the Isoflavone reductase gene in Bulbophyllum and other orchids

Bulbophyllum, the largest genus in Orchidaceae, is known for being rich in phenolic compounds. In this study, the polyphenol and antioxidant content of four Bulbophyllum species was compared to that of a Dendrobium sp. control. The transcript levels of eight genes involved in flavonoid biosynthesis and oxidative stress responses were then determined to identify correlations with flavonoid and antioxidant content. A positive correlation between flavonoid content and transcription level of isoflavone reductase (IFR), belonging to the short-chain dehydrogenase/reductase (SDR) superfamily, was observed in leaf and pseudobulb tissue of the orchids. IFR showed an extraordinarily high level of gene expression (max. 118-fold) in the pseudobulb tissue of Bulbophyllum species compared with that in the same tissue in several species of Dendrobium. A pair of primers derived from two conserved regions within the sequences of related SDRs (IFR, eugenol synthase, and phenylcoumaran benzylic ether reductase) from Dendrobium spp., Paphiopedilum sp., Phaseolus sp., Phoenix sp., Striga sp., and Vanda sp. were used to attain a full-length IFR from four Bulbophyllum spp., three Dendrobium spp. and one Vanda sp. The phylogenetic relationships of those SDRs reported in the NCBI database showed distinctive clusters between the closely related SDRs of each genus in the Orchidaceae, and the distantly related clusters of the other plant SDRs. This work describes an easy method for the quick detection and isolation of IFRs in Bulbophyllum and other orchid species.

Comparative proteome analyses of rhizomania resistant transgenic sugar beets based on RNA silencing mechanism

ABSTRACT Rhizomania is an economically important disease of sugar beet, which is caused by Beet necrotic yellow vein virus (BNYVV). As previously shown, RNA silencing mechanism effectively inhibit the viral propagation in transgenic sugar beet plants. To investigate possible proteomic changes induced by gene insertion and/or RNA silencing mechanism, the root protein profiles of wild type sugar beet genotype 9597, as a control, and transgenic events named 6018-T3:S6-44 (S6) and 219-T3:S3-13.2 (S3) were compared by two-dimensional gel electrophoresis. The accumulation levels of 25 and 24 proteins were differentially regulated in S3 and S6 plants, respectively. The accumulation of 15 spots were increased or decreased more than 2-fold. Additionally, 10 spots repressed or induced in both, while seven spots showed variable results in two events. All the differentially expressed spots were analyzed by MALDI-TOF-TOF mass spectrometry. The functional analysis of differentially accumulated proteins showed that most of them are related to the metabolism and defense/stress response. None of these recognized proteins were allergens or toxic proteins except for a spot identified as phenylcoumaran benzylic ether reductase, Pyrc5, which was decreased in the genetically modified S6 plant. These data are in favor of substantial equivalence of the transgenic plants in comparison to their related wild type cultivar since the proteomic profile of sugar beet root was not remarkably affected by gene transfer and activation RNA silencing mechanism.

Expression and functional analyses of a putative phenylcoumaran benzylic ether reductase in Arabidopsis thaliana

A candidate gene for phenylcoumaran benzylic ether reductase in Arabidopsis thaliana encodes a peptide with predicted functional activity and plays a crucial role in secondary metabolism. Phenylcoumaran benzylic ether reductase (PCBER) is thought to be an enzyme crucial in the biosynthesis of 8-5'-linked neolignans. Genes of the enzyme have been isolated and characterized in several plant species. In this study, we cloned cDNA and the 5'-untranslated region of one PCBER candidate gene (At4g39230, designated AtPCBER1) from Arabidopsis thaliana. At the amino acid level, AtPCBER1 shows high sequence identity (64-71 %) with PCBERs identified from other plant species. Expression analyses of AtPCBER1 by reverse transcriptase-polymerase chain reaction and histochemical analysis of transgenic plants harboring the 5'-untranslated region of AtPCBER1 linked with gus coding sequence indicate that expression is induced by wounding and is expressed in most tissues, including flower, stem, leaf, and root. Catalytic analysis of recombinant AtPCBER1 with neolignan and lignans in the presence of NADPH suggests that the protein can reduce not only the 8-5'-linked neolignan, dehydrodiconiferyl alcohol, but also 8-8' linked lignans, pinoresinol, and lariciresinol, with lower activities. To investigate further, we performed metabolomic analyses of transgenic plants in which the target gene was up- or down-regulated. Our results indicate no significant effects of AtPCBER1 gene regulation on plant growth and development; however, levels of some secondary metabolites, including lignans, flavonoids, and glucosinolates, differ between wild-type and transgenic plants. Taken together, our findings indicate that AtPCBER1 encodes a polypeptide with PCBER activity and has a critical role in the biosynthesis of secondary metabolites in A. thaliana.

Relative expression of seven candidate genes for pathogen resistance on Pinus radiata infected with Fusarium circinatum

The objective of this research was to assess the relative expression of candidate genes associated to plant disease resistance in Pinus radiata inoculated with Fusarium circinatum by using two different inoculation methods. Microdroplet inoculation allowed an earlier and reliable selection of contrasting genotypes, however, spray inoculation method was more informative for gene expression analysis. Thaumatin-like protein, peroxidase, phenylalanine ammonia lyase, pinosylvin synthase and phenylcoumaran benzylic ether reductase genes increased significantly their transcription levels after inoculation with the pathogen in the resistant genotype, while in the susceptible genotype these genes showed lower relative expression.

Phenylcoumaran benzylic ether reductase prevents accumulation of compounds formed under oxidative conditions in poplar xylem.

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8-5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.

Structural functionality, catalytic mechanism modeling and molecular allergenicity of phenylcoumaran benzylic ether reductase, an olive pollen (Ole e 12) allergen

Isoflavone reductase-like proteins (IRLs) are enzymes with key roles in the metabolism of diverse flavonoids. Last identified olive pollen allergen (Ole e 12) is an IRL relevant for allergy amelioration, since it exhibits high prevalence among atopic patients. The goals of this study are the characterization of (A) the structural-functionality of Ole e 12 with a focus in its catalytic mechanism, and (B) its molecular allergenicity by extensive analysis using different molecular computer-aided approaches covering (1) physicochemical properties and functional-regulatory motifs, (2) sequence analysis, 2-D and 3D structural homology modeling comparative study and molecular docking, (3) conservational and evolutionary analysis, (4) catalytic mechanism modeling, and (5) sequence, structure-docking based B-cell epitopes prediction, while T-cell epitopes were predicted by inhibitory concentration and binding score methods. Structural-based detailed features, phylogenetic and sequences analysis have identified Ole e 12 as phenylcoumaran benzylic ether reductase. A catalytic mechanism has been proposed for Ole e 12 which display Lys133 as one of the conserved residues of the IRLs catalytic tetrad (Asn-Ser-Tyr-Lys). Structure characterization revealed a conserved protein folding among plants IRLs. However, sequence polymorphism significantly affected residues involved in the catalytic pocket structure and environment (cofactor and substrate interaction-recognition). It might also be responsible for IRLs isoforms functionality and regulation, since micro-heterogeneities affected physicochemical and posttranslational motifs. This polymorphism might have large implications for molecular differences in B- and T-cells epitopes of Ole e 12, and its identification may help designing strategies to improve the component-resolving diagnosis and immunotherapy of pollen and food allergy through development of molecular tools.

Natural hypolignification is associated with extensive oligolignol accumulation in flax stems.

Flax (Linum usitatissimum) stems contain cells showing contrasting cell wall structure: lignified in inner stem xylem tissue and hypolignified in outer stem bast fibers. We hypothesized that stem hypolignification should be associated with extensive phenolic accumulation and used metabolomics and transcriptomics to characterize these two tissues. (1)H nuclear magnetic resonance clearly distinguished inner and outer stem tissues and identified different primary and secondary metabolites, including coniferin and p-coumaryl alcohol glucoside. Ultrahigh-performance liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry aromatic profiling (lignomics) identified 81 phenolic compounds, of which 65 were identified, to our knowledge, for the first time in flax and 11 for the first time in higher plants. Both aglycone forms and glycosides of monolignols, lignin oligomers, and (neo)lignans were identified in both inner and outer stem tissues, with a preponderance of glycosides in the hypolignified outer stem, indicating the existence of a complex monolignol metabolism. The presence of coniferin-containing secondary metabolites suggested that coniferyl alcohol, in addition to being used in lignin and (neo)lignan formation, was also utilized in a third, partially uncharacterized metabolic pathway. Hypolignification of bast fibers in outer stem tissues was correlated with the low transcript abundance of monolignol biosynthetic genes, laccase genes, and certain peroxidase genes, suggesting that flax hypolignification is transcriptionally regulated. Transcripts of the key lignan genes Pinoresinol-Lariciresinol Reductase and Phenylcoumaran Benzylic Ether Reductase were also highly abundant in flax inner stem tissues. Expression profiling allowed the identification of NAC (NAM, ATAF1/2, CUC2) and MYB transcription factors that are likely involved in regulating both monolignol production and polymerization as well as (neo)lignan production.

Crystal Structure and Catalytic Mechanism of Leucoanthocyanidin Reductase from Vitis vinifera

Leucoanthocyanidin reductase (LAR) catalyzes the NADPH-dependent reduction of 2 R,3 S,4 S-flavan-3,4-diols into 2 R,3 S-flavan-3-ols, a subfamily of flavonoids that is important for plant survival and for human nutrition. LAR1 from Vitis vinifera has been co-crystallized with or without NADPH and one of its natural products, (+)-catechin. Crystals diffract to a resolution between 1.75 and 2.72 Å. The coenzyme and substrate binding pocket is preformed in the apoprotein and not markedly altered upon NADPH binding. The structure of the abortive ternary complex, determined at a resolution of 2.28 Å, indicates the ordering of a short 3 10 helix associated with substrate binding and suggests that His122 and Lys140 act as acid–base catalysts. Based on our 3D structures, a two-step catalytic mechanism is proposed, in which a concerted dehydration precedes an NADPH-mediated hydride transfer at C4. The dehydration step involves a Lys-catalyzed deprotonation of the phenolic OH7 through a bridging water molecule and a His-catalyzed protonation of the benzylic hydroxyl at C4. The resulting quinone methide serves as an electrophilic target for hydride transfer at C4. LAR belongs to the short-chain dehydrogenase/reductase superfamily and to the PIP (pinoresinol–lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase) family. Our data support the concept that all PIP enzymes reduce a quinone methide intermediate and that the major role of the only residue that has been conserved from the short-chain dehydrogenase/reductase catalytic triad (Ser…TyrXXXLys), that is, lysine, is to promote the formation of this intermediate by catalyzing the deprotonation of a phenolic hydroxyl. For some PIP enzymes, this lysine-catalyzed proton abstraction may be sufficient to trigger the extrusion of the leaving group, whereas in LAR, the extrusion of a hydroxide group requires a more sophisticated mechanism of concerted acid–base catalysis that involves histidine and takes advantage of the OH4, OH5, and OH7 substituents of leucoanthocyanidins.

Analysis of expressed sequence tags from Ginkgo mature foliage in China

Ginkgo biloba L. is a tree native to China, which has large importance within medicine and horticulture. The extracts from Ginkgo mature leaves with rich flavonoids and terpenoids are commonly used for a variety of folk remedies. We constructed a cDNA library derived from mature leaves of Ginkgo, which consisted of 8.12 × 105 clones with the insert length of 500–2,000 bp. We performed an analysis of expressed sequence tags (ESTs) and obtained partial sequences from 2,039 clones, which represented 1,437 unigenes consisting of 249 contigs and 1,188 singletons. The 2,039 ESTs were submitted to GenBank (dbEST) at NCBI and were assigned GenBank accession numbers from GE647881 to GE649919. The 1,235 cDNA clones out of 2,039 (60.1%) were assigned putative functions, and the remaining 804 clones were not similar to any known gene sequences in the databases. The five largest categories of Ginkgo clones were: “energy” (19.4%), “disease/defense” (16%), “metabolism” (11.3%), “unclassified proteins” (12.5%), and "secondary metabolism" (9%). The highly expressed transcripts in the cDNA library were some genes related to photosynthesis, disease/defense, and flavonoid biosynthesis, including ribulose–bisphosphate carboxylase small-chain gene, pathogenesis-related protein gene, light-harvesting chlorophyll a/b binding protein of photosystem gene, catalase gene, and phenylcoumaran benzylic ether reductase gene et al. Many genes with ESTs similar to photosynthesis, secondary metabolism, and stress-response genes were characterized. The analysis of ESTs indicates that it is a useful approach for isolating Ginkgo genes homologous to known genes. Our results provide new information about mature leaf-specific transcripts of Ginkgo.

Expression of a phenylcoumaran benzylic ether reductase-like protein in the ovules of Gossypium hirsutum

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to identify differentially expressed proteins in wild-type (DP 5690) and fiberless (SL 1-7-1) cotton ovules. One protein, designated V2 was unique to ovules of the fiber producing DP 5690 line. The protein was purified from 2D-PAGE of 4 d post anthesis DP 5690 ovules and partially sequenced. The short amino acid sequence was nearly identical to the deduced amino acid sequence for cotton phenylcoumaran benzylic ether reductase (PCBER) protein. A consensus sequence was assembled from ESTs encoding cotton PCBER genes, primers were designed, and a full length gene was amplified from plasmid DNA from a 72 h etiolated cotton cotyledon library. The polymerase chain reaction generated a 950 bp product with unique EcoRI (5′) and (3′) KpnI restriction sites for directional insertion into the expression vector pPICZA. Nucleotide sequencing was performed, and the full length coding region was 924 bp encoding a protein of 308 amino acids. The molecular mass and pI measured (2D PAGE) were similar to the theoretical protein.

Hinokinin biosynthesis in Linum corymbulosum Reichenb.

Due to their peculiar stereochemistry and numerous biological activities, lignans are of widespread interest. As only a few biosynthetic steps have been clarified to date, we aimed to further resolve the molecular basis of lignan biosynthesis. To this end, we first established that the biologically active lignan (-)-hinokinin could be isolated from in vitro cultures of Linum corymbulosum. Two hypothetical pathways were outlined for the biosynthesis of (-)-hinokinin. In both pathways, (+)-pinoresinol serves as the primary substrate. In the first pathway, pinoresinol is reduced via lariciresinol to secoisolariciresinol by a pinoresinol-lariciresinol reductase, and methylenedioxy bridges are formed later. In the second pathway, pinoresinol itself is the substrate for formation of the methylenedioxy bridges, resulting in consecutive production of piperitol and sesamin. To determine which of the proposed hypothetical pathways acts in vivo, we first isolated several cDNAs encoding one pinoresinol-lariciresinol reductase (PLR-Lc1), two phenylcoumaran benzylic ether reductases (PCBER-Lc1 and PCBER-Lc2), and two PCBER-like proteins from a cDNA library of L. corymbulosum. PLR-Lc1 was found to be enantiospecific for the conversion of (+)-pinoresinol to (-)-secoisolariciresinol, which can be further converted to give (-)-hinokinin. Hairy root lines with significantly reduced expression levels of the plr-Lc1 gene were established using RNAi technology. Hinokinin accumulation was reduced to non-detectable levels in these lines. Our results strongly indicate that PLR-Lc1 participates in (-)-hinokinin biosynthesis in L. corymbulosum by the first of the two hypothetical pathways via (-)-secoisolariciresinol.

Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining

Isoflavans and pterocarpans are the major biosynthetically connected phytoalexins in legumes. A search of the expressed sequence tag library of a model legume Lotus japonicus, which produces an (−)-isoflavan, for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans, yielded seven full-length cDNAs, and the encoded proteins were analyzed in vitro. Four of them cleaved the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (−)-vestitol and were designated pterocarpan reductase (PTR). Two PTRs displayed enantiospecificity to (−)-medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types.

Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester

Phenylpropenes such as chavicol, t-anol, eugenol, and isoeugenol are produced by plants as defense compounds against animals and microorganisms and as floral attractants of pollinators. Moreover, humans have used phenylpropenes since antiquity for food preservation and flavoring and as medicinal agents. Previous research suggested that the phenylpropenes are synthesized in plants from substituted phenylpropenols, although the identity of the enzymes and the nature of the reaction mechanism involved in this transformation have remained obscure. We show here that glandular trichomes of sweet basil (Ocimum basilicum), which synthesize and accumulate phenylpropenes, possess an enzyme that can use coniferyl acetate and NADPH to form eugenol. Petunia (Petunia hybrida cv. Mitchell) flowers, which emit large amounts of isoeugenol, possess an enzyme homologous to the basil eugenol-forming enzyme that also uses coniferyl acetate and NADPH as substrates but catalyzes the formation of isoeugenol. The basil and petunia phenylpropene-forming enzymes belong to a structural family of NADPH-dependent reductases that also includes pinoresinol-lariciresinol reductase, isoflavone reductase, and phenylcoumaran benzylic ether reductase.

Identification and Characterization of Cor33p, a Novel Protein Implicated in Tolerance towards Oxidative Stress inCandida albicans

We applied two-dimensional gel electrophoresis to identify downstream effectors of CPH1 and EFG1 under hypha-inducing conditions in Candida albicans. Among the proteins that were expressed in wild-type cells but were strongly downregulated in a cph1Delta/efg1Delta double mutant in alpha-minimal essential medium at 37 degrees C, we could identify not-yet-characterized proteins, including Cor33-1p and Cor33-2p. The two proteins are almost identical (97% identity) and represent products of allelic isoforms of the same gene. Cor33p is highly similar to Cip1p from Candida sp. but lacks any significant homology to proteins from Saccharomyces cerevisiae. Strikingly, both proteins share homology with phenylcoumaran benzylic ether reductases and isoflavone reductases from plants. For other hypha-inducing media, like yeast-peptone-dextrose (YPD) plus serum at 37 degrees C, we could not detect any transcription for COR33 in wild-type cells, indicating that Cor33p is not hypha specific. In contrast, we found a strong induction for COR33 when cells were treated with 5 mM hydrogen peroxide. However, under oxidative conditions, transcription of COR33 was not dependent on EFG1, indicating that other regulatory factors are involved. In fact, upregulation depends on CAP1 at least, as transcript levels were clearly reduced in a Deltacap1 mutant strain under oxidative conditions. Unlike in wild-type cells, transcription of COR33 in a tsa1Delta mutant can be induced by treatment with 0.1 mM hydrogen peroxide. This suggests a functional link between COR33 and thiol-specific antioxidant-like proteins that are important in the oxidative-stress response in yeasts. Concordantly, cor33Delta deletion mutants show retarded growth on YPD plates supplemented with hydrogen peroxide, indicating that COR33 in general is implicated in conferring tolerance toward oxidative stress on Candida albicans.

Occupational asthma due to carrot in a cook

Background Few previous reports of carrot-induced asthma have been confirmed by objective tests. Hypersensitivity to carrot is frequently associated with allergy to Apiaceae spices and sensitization to birch and mugwort pollens. Clinical case A 40-year-old cook woman was seen with sneezing, rhinorrhea, contact urticaria and wheezing within few minutes of handling or cutting raw carrots. She needed to leave out of the kitchen while the other cooks cut raw carrots. Methods and results Skin tests were positive to carrot, celery, aniseed and fennel. Rubbing test with fresh carrot was positive. Specific IgE to carrot was 4.44 kU/L. Determinations of specific IgE to mugwort, grass and birch pollens were negative. Inhalative provocation test, performed as a handling test, was positive. The IgE-immunoblotting showed two bands in carrot extract: a band with apparent molecular weight of 30 kd and other band of 18 kd. This band of 18 kd was Dau c 1. The band of 30 kd could correspond a phenylcoumaran benzylic ether reductase. Dau c 1 did not appear to be the unique allergen in this case. Additional allergens may induce the sensitization. Primary sensitization due to airborne allergens of foods and the lack of pollen allergy in this patient are notorious events.

A new latex allergen classified into the plant defense-related reductase family

Rationale As sequence information on genes and proteins is accumulated in databases, it becomes easy to identify a large number of proteins in a short time with proteomics approaches. Application of such techniques to allergenic proteins is referred to allergenomics. In this study, we tried to find unidentified latex allergens with an allergenomics method. Methods Proteins extracted from non-ammoniated rubber latex was two-dimensionally separated according to their isoelectric points (pI) and molecular weights then transferred onto a membrane. IgE-recognizing proteins were visualized by immunoblotting using a pooled serum from latex-allergic people. Spots of the allergenic proteins were cut out form the gel and the allergens were site-specifically in-gel digested by trypsin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) spectra of the resultant fragments and MS/MS spectra of some prominent peptide-ions were recorded on a TOF-TOF instrument. From these mass-spectrometric data, the allergens were assigned by database searches. Results A few new latex allergens as well as several previously reported allergens such as patatin-like proteins (Hev b 7), enolase (Hev b 9), and havamine were identified with this approach. One of the novel latex allergens (32 kD pI 5.2) was sequentially homologous to plant defense-related reductases like isoflavone reductase and phenylcoumaran benzylic ether reductase. Conclusions We first identified a defense-related reductase as a latex allergen. Several allergens belonging to the plant defense-related reductase family (Bet v 6-related allergens) have already been found from fruits, vegetables, and pollen. Considering the sequence similarities among them, we can predict the cross-reactivity of the defense-related reductase found from natural rubber.

Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

An historical perspective on lignan biosynthesis: Monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism

This review describes discoveries from this laboratory on monolignol, allylphenol and hydroxycinnamic acid coupling, and downstream metabolic conversions, affording various lignan skeleta. Stereoselective 8-8′ coupling (dirigent protein-mediated) of coniferyl alcohol to afford (+)-pinoresinol is comprehensively discussed, as is our current mechanistic/kinetic understanding of the protein’s radical-radical binding, orientation and coupling properties, and insights gained for other coupling modes, e.g. affording (−)-pinoresinol. In a species dependent manner, (+)- or (−)-pinoresinols can also undergo enantiospecific reductions, catalyzed by various bifunctional pinoresinol-lariciresinol reductases (PLR), to afford lariciresinol and then secoisolariciresinol. With X-ray structures giving a molecular basis for differing PLR enantiospecificities, comparisons are made herein to the X-ray structure of the related enzyme, phenylcoumaran benzylic ether reductase, capable of 8-5′ linked lignan regiospecific reductions. Properties of the enantiospecific secoisolariciresinol dehydrogenase (also discovered in our laboratory and generating 8-8′ linked matairesinol) are summarized, as are both in situ hybridization and immunolocalization of lignan pathway mRNA/proteins in vascular tissues. This entire 8-8′ pathway thus overall affords secoisolariciresinol and matairesinol, viewed as cancer preventative agent precursors, as well as intermediates to cancer treating substances, such as podophyllotoxin derivatives. Another emphasis is placed on allylphenol/hydroxycinnamic acid coupling and associated downstream metabolism, e.g. affording the antiviral creosote bush lignan, nordihydroguaiaretic acid (NDGA), and the fern lignans, blechnic/brainic acids. Regiospecific 8-8′ allylphenol coupling is described, as is characterization of the first enantiospecific membrane-bound polyphenol oxidase, (+)-larreatricin hydroxylase, involved in NDGA formation. Specific [13C]-labeling also indicated that Blechnum lignans arise from stereoselective 8-2′ hydroxycinnamic acid coupling. Abbreviations: CD – circular dichroism; e.e. – enantiomeric excess; DP – dirigent protein; ESI-MS – electrospray ionization mass spectrometry; MALDI -TOF – matrix assisted laser desorption ionization-time of flight; MALLS – multiangle laser light scattering; PLR – pinoresinol lariciresinol reductase; SDH – secoisolariciresinol dehydrogenase.

Expression patterns of two tobacco isoflavone reductase-like genes and their possible roles in secondary metabolism in tobacco.

Plants contain a large number of proteins homologous to isoflavone reductase, an NADPH-dependent reductase involved in the biosynthesis of isoflavonoid phytoalexins in legumes. Although some are bonafide isoflavone reductases, others may catalyze distinct reductase reactions. Two tobacco genes, TP7 and A622, encoding isoflavone reductase-like proteins, had been previously identified from their unique expression patterns, but their functions were not known. We show here that TP7 is a tobacco phenylcoumaran benzylic ether reductase involved in lignan biosynthesis, but that A622 is not. To gain insight into the possible function of A622, we analyzed in detail the expression patterns of the A622 gene by RNA and protein blots, immunohistochemistry, and its promoter expression in transgenic Nicotiana sylvestris roots. The A622 expression patterns were qualitatively similar to those of putrescine N-methyltransferase, the first enzyme in nicotine biosynthesis, suggesting that A622 may function in the metabolism of nicotine or related alkaloids.