Opposite Stereoselectivities of Dirigent Proteins in Arabidopsis and Schizandra Species

How stereoselective monolignol-derived phenoxy radical-radical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (−)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (−)-pinoresinol-forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidase reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (−)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (−)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinol-forming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes.     

Expression in Pichia pastoris and characterization of two novel dirigent proteins for atropselective formation of gossypol

We established an efficient fed-batch fermentation process for two novel dirigent proteins from cotton plants, GbDIR2 from Gossypium barbadense and GhDIR3 from G. hirsutum, using the engineered Pichia pastoris GlycoSwitch® SuperMan₅ strain to prevent hyperglycosylation. The two (His)₆-tagged proteins were purified by metal-chelate affinity chromatography and obtained in quantities of 12 and 15 mg L⁻¹ of culture volume, respectively. Glycosylation sites were identified for the native and for the enzymatically deglycosylated proteins by mass spectrometry, confirming five to six of the seven predicted glycosylation sites in the NxS/T sequence context. The predominant glycan structure was Man₅GlcNAc₂ with, however, a significant contribution of Man_4–10GlcNAc₂. Both dirigent proteins (DIRs) mediated the formation of (+)-gossypol by atropselective coupling of hemigossypol radicals. Similar to previously characterized DIRs, GbDIR2 and GhDIR3 lacked oxidizing activity and depended on an oxidizing system (laccase/O₂) for the generation of substrate radicals. In contrast to DIRs involved in the biosynthesis of lignans, glycosylation was not essential for function. Quantitative enzymatic deglycosylation yielded active GbDIR2 and GhDIR3 in excellent purity. The described fermentation process in combination with enzymatic deglycosylation will pave the way for mechanistic and structural studies and, eventually, the application of cotton DIRs in a biomimetic approach towards atropselective biaryl synthesis.

     

COX,LOX and platelet aggregation inhibitory properties of Lauraceae neolignans

The anti-inflammatory potential of 26 neolignans (14 of the bicyclooctane-type and 12 of the benzofuran-type), isolated from three Lauraceae species (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), was evaluated in vitro through inhibition of COX-1, COX-2, 5-LOX and agonist-induced aggregation of rabbit platelets. Benzofuran neolignans were found to be selective COX-2 inhibitors, whereas bicyclooctane neolignans inhibit selectively the PAF-action as well as COX-1 and 5-LOX. The neolignan 9-nor-7,8-dehydro-isolicarin B 15 and cinerin C 7 were found to be the most potent COX-2 inhibitor and PAF-antagonist, respectively. Nectamazin C 10 exhibited dual 5-LOX/COX-2 inhibition.     

Pleiotropic changes in Arabidopsis <Emphasis Type="Italic">f5h</Emphasis> and <Emphasis Type="Italic">sct</Emphasis> mutants revealed by large-scale gene expression and metabolite analysis

Hydrocinnamic acid esters, lignin, flavonoids, glucosinolates, and salicylic acid protect plants against UV exposure, oxidative stress, diseases, and herbivores. Through the phenylpropanoid pathway, certain Brassicaceae family members, including Arabidopsis thaliana and Brassica napus, accumulate large amounts of the anti-nutritive sinapoylcholine (sinapine) in the seed. We successfully down-regulated activities of key enzymes in the pathway including F5H and SCT and achieved reduction of sinapine and lignin in B. napus seeds. Despite this success, it was unclear how multiple agronomic traits were affected in the transgenic plants. Here, we report altered large-scale gene expression of new alleles of f5h and sct mutants of A. thaliana and resultant accumulation of sinapoylglucose, disinapoylglucose, quercetin-3-O-rhamnoside, salicylic acid glucoside, and total indolyl glucosinolates in the two mutants. Expression of several flowering genes was altered in these mutants when grown under drought and NaCl treatments. Furthermore, both mutants were more susceptible to fungal infection than the wild type. Microarray experiments identified distinctive spatial and temporal expression patterns of gene clusters involved in silique/seed developmental processes and metabolite biosynthesis in these mutants. Taken together, these findings suggest that both f5h and sct mutants exhibit major differences in accumulation of diverse metabolites in the seed and profound changes in global large-scale gene expression, resulting in differential pleiotropic responses to environmental cues.     

Further lignoids from Virola sebifera

From the fruits of Virola sebifera 2,3-dibenzylbutanes were isolated together with tetralin and naphthalene neolignans. 2,3-Dimethyl-4-piperonyl-4-veratrylbutan-1-ol, a further constituent, probably arises by a retro-Friedel-Crafts reaction from a tetralin neolignan. An additional component was identified as a dimeric neolignan.     

Trimeric Structure of (+)-Pinoresinol-forming Dirigent Protein at 1.95 ? Resolution with Three Isolated Active Sites

Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (−)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.     

Neolignans from the seeds of Prunus tomentosa (Rosaceae) and their chemotaxonomic interest

The role of neolignans in the chemotaxonomy of the genus Prunus has long been neglected. In this study, two new dihydrobenzofuran neolignans tomentosanans A (12) and B (14), together with 15 known neolignans, were isolated from the seeds of Prunus tomentosa. This is the first report on the occurrence of compounds 1–12, 14, 15 and 17 in the genus Prunus. The biosynthetic pathways of these neolignans are also summarized. Together, these neolignans represent a phytochemical fingerprint for P. tomentosa, which may be used for identification of Prunus species.     

RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: Consequences on lignans and neolignans accumulation

RNAi technology was applied to down regulate LuPLR1 gene expression in flax (Linum usitatissimum L.) seeds. This gene encodes a pinoresinol lariciresinol reductase responsible for the synthesis of (+)-secoisolariciresinol diglucoside (SDG), the major lignan accumulated in the seed coat. If flax lignans biological properties and health benefits are well documented their roles in planta remain unclear. This loss of function strategy was developed to better understand the implication of the PLR1 enzyme in the lignan biosynthetic pathway and to provide new insights on the functions of these compounds. RNAi plants generated exhibited LuPLR1 gene silencing as demonstrated by quantitative RT-PCR experiments and the failed to accumulate SDG. The accumulation of pinoresinol the substrate of the PLR1 enzyme under its diglucosylated form (PDG) was increased in transgenic seeds but did not compensate the overall loss of SDG. The monolignol flux was also deviated through the synthesis of 8-5′ linked neolignans dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) which were observed for the first time in flax seeds.     

Two new dihydrobenzofuran-type neolignans from Breynia fruticosa

(7S,8R,7′S)-9,7′,9′-Trihydroxy-3,4-methylenedioxy-3′-methoxy [7-O-4′,8-5′] neolignan (1) and (7S,8R,7′S)-9,9′-dihydroxy-3,4-methylenedioxy-3′,7′-dimethoxy [7-O-4′,8-5′] neolignan (2), two new natural dihydrobenzofuran-type neolignans, along with 9,9′-dihydroxy-3,4-methylenedioxy-3′-methoxy [7-O-4′,8-5′] neolignan (3) and (-)-machicendiol (4), were isolated from the whole plants of Breynia fruticosa. The structures of 1 and 2, including the absolute configurations, were determined by spectroscopic methods and circular dichroism (CD) techniques. The absolute configuration of 4 was confirmed by calculations of the OR spectrum, together with OR and ECD spectra of its p-bromobenzoate ester (4a).     

Ethylene signaling may be involved in the regulation of tocopherol biosynthesis in Arabidopsis thaliana

Tocopherol biosynthesis was investigated in ein3-1, etr1-1 and eto1-1 mutants of Arabidopsis thaliana, which show a defect in ethylene signaling, perception and over-produce ethylene, respectively. A mutation in the EIN3 gene delayed the water-stress related increase in α-tocopherol and caused a reduction in the levels of this antioxidant by ca. 30% compared to the wild type. In contrast to the wild type and ein3-1 mutants, both etr1-1 and eto1-1 mutants showed a sharp (up to 5-fold) increase in α-tocopherol levels during leaf aging. It is concluded that ethylene perception and signaling may be involved in the regulation of tocopherol biosynthesis during water stress and leaf aging.     

DGAT1 and PDAT1 Acyltransferases Have Overlapping Functions in Arabidopsis Triacylglycerol Biosynthesis and Are Essential for Normal Pollen and Seed Development

Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.     

Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2′-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2′-methylthio)ethylmalate ∼100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.     

Eusiderins and other neolignans from an Aniba species

A previous report disclosed the presence of benzodioxan and bicyclo[3.2.1]octanoid neolignans in the benzene extract of the trunk wood of an Amazonian Aniba (Lauraceae) species. The chloroform extract of the same material contains additionally two new benzodioxan neolignans [rel-(7S,8R)-Δ^8′-7-hydroxy-3,4,5,5′-tetramethoxy-7.0.3′,8.0.4′-neolignan; rel-(7R,8R)-Δ^7′-3,4,5,5′-tetramethoxy-9′-oxo-7.0.3′,8.0.4′-neolignan], two new bicyclo[3.2.1]-octanoid neolignans [(7R,8S,1′S,2′S,3′S,4′R)-Δ^8′-2′,4′-dihydroxy-3,3′-dimethoxy-4,5-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan; (7R,8S,1′R,2′S,3′S)-Δ^8′-2′-hydroxy-3,3′,5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-4′-oxo-7.3′,8.1′-neolignan] and a hydrobenzofuranoid neolignan [(7S,8R,1′S,5′S)-Δ^8′-3,3′,5′-tri-methoxy-4,5-methylenedioxy-1′,4′,5′,6′-tetrahydro-4′-oxo-7.0.2′,8.1-neolignan].     

Phosphoenolpyruvate Provision to Plastids Is Essential for Gametophyte and Sporophyte Development in Arabidopsis thaliana

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1(+/−)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/−) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/−) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis.     

in Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1)

Cinnamoyl CoA reductase (CCR) carries out the first committed step in monolignol biosynthesis and acts as a first regulatory point in lignin formation. CCR shows multiple substrate specificity towards various cinnamoyl CoA esters. Here, in Silico mutagenesis studies of active site residues of Ll-CCRH1 were carried out. Homology modeling based modeled 3D structure of Ll-CCRH1 was used as template for in Silico mutant preparations. Docking simulations of Ll-CCRH1 mutants with CoA esters by AutoDock Vina tools showed altered substrate specificity as compared to wild type. The study evidences that conformational changes, and change in geometry or architecture of active site pocket occurred following mutations. The altered substrate specificity for active site mutants suggests the possible physiological role of CCR either in lignin formation or in defense system in plants.

Abbreviations

Ll-CCRH1 - Leucaena leucocephala cinnamoyl CoA reductase 1, OPLS - Optimized Potentials for Liquid Simulations, RMSD - Root Mean Square Deviation.     

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3′-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3′H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide–binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP⁺ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.