Peroxisomal 4-coumaroyl-CoA ligases participate in shikonin production in Lithospermum erythrorhizon.

4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: class I 4CLs involved in lignin metabolism, class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least eight 4CL genes, among which the expression of three (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from class I and class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the three Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We therefore conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.

Novel ability of diflubenzuron as an inhibitor of mitochondrial function.

Compounds classified as benzoylphenylurea (BPU), such as diflubenzuron (DFB), are used as insecticides. Although BPU disrupts molting by inhibiting chitin biosynthesis and exhibits insecticidal activity, their exact mode of action remains unknown. Since epidermal cells proliferate and morphologically change from squamous to columnar cells during the early stages of insect molting, we speculate that a transition similar to that from epithelium to mesenchyme occurs and that BPU may inhibit this transition. Here, we addressed this possibility. We found that DFB decreases actin expression in insect cells (the tissue cultures of insect integument). Detailed analysis in Schneider S2 cells reveals that DFB inhibits the expression of actin isoforms (Act5C and Act42A) and the Drosophila ortholog of myocardin-related transcription factor (Mrtf), leading to cell growth suppression. Proteomics identified the Drosophila ortholog of prohibitin (Phb1D and Phb2E) as one of the DFB-binding proteins. DFB inhibits the interaction between Phb1D and Phb2E and induces mitochondrial dysfunction. The knock-down of Phb2E suppresses the expression of Act5C, Act42A, and Mrtf, leading to cell growth inhibition. Thus, the disruption of Phb function is a possible novel target of DFB.

Quantitative evaluation of the biological activity of various brassinosteroids using spiral root induction in rice seeds.

Spiral roots are induced in germinated rice seeds through treatment with nanomolar brassinosteroids (BRs) but not with other plant hormones. Here, we determined the minimum effective concentration (MEC) of various BRs to induce spiral roots in germinated rice seeds. The reciprocal logarithm of MEC, pMEC, was used as the BL-like activity index, which was linearly correlated with the reciprocal logarithm of a 50% effective dose (pED50) as evaluated in the rice lamina inclination assay. Furthermore, a ligand-receptor docking simulation was performed against the BL receptor complex, Arabidopsis thaliana BRI1/SERK1, and the binding free energy (ΔGbind) was calculated for the tested BRs. The ΔGbind calculation was performed using the molecular mechanics/generalized Born surface area method on an ensemble of uncorrelated snapshots collected via molecular dynamics to predict biological activity.

Structure-activity relationship of volatile compounds that induce defense-related genes in maize seedlings.

Volatile organic compounds mediate plant-to-plant communication, and plants receiving volatile cues can acquire greater defenses against attackers. It has been expected that volatiles are received by factors that eventually lead to the induction of defense-related gene expression; however, the nature of these factors remain unclear. Structure-activity relationship analysis of gene expression induction by volatiles should provide insights into the nature of these factors. We conducted a structure-activity relationship study using maize seedlings and (Z)-3-hexen-1-yl acetate (Z3HAC) as the lead compound. The acid portion of Z3HAC was not essential, and (Z)-3-hexen-1-ol (Z3HOL), which is formed after the hydrolysis of Z3HAC, is likely the structure essential for the upregulation of the genes. The double bond of Z3HOL is essential; however, its geometry is indistinguishable. Strict specificity was detected regarding the length of the methylene chain on the α- and ω-sides of the double bond, and therefore, the 3-hexen-1-ol structure was found to be the ultimate structure. This finding provides insight into the nature of the factors that interact with a volatile compound and subsequently activate signaling pathways, leading to the upregulation of a subset of defense genes.

DIENELACTONE HYDROLASE LIKE PROTEIN1 negatively regulates the KAI2-ligand pathway in Marchantia polymorpha.

Karrikins are smoke-derived butenolides that induce seed germination and photomorphogenesis in a wide range of plants.1,2,3 KARRIKIN INSENSITIVE2 (KAI2), a paralog of a strigolactone receptor, perceives karrikins or their metabolized products in Arabidopsis thaliana.4,5,6,7 Furthermore, KAI2 is thought to perceive an unidentified plant hormone, called KAI2 ligand (KL).8,9 KL signal is transduced via the interaction between KAI2, MORE AXILLARY GROWTH2 (MAX2), and SUPPRESSOR of MORE AXILLARY GROWTH2 1 LIKE family proteins (SMXLs), followed by the degradation of SMXLs.4,7,10,11,12,13,14 This signaling pathway is conserved both in A. thaliana and the bryophyte Marchantia polymorpha.14 Although the KL signaling pathway is well characterized, the KL metabolism pathways remain poorly understood. Here, we show that DIENELACTONE HYDROLASE LIKE PROTEIN1 (DLP1) is a negative regulator of the KL pathway in M. polymorpha. The KL signal induces DLP1 expression. DLP1 overexpression lines phenocopied the Mpkai2a and Mpmax2 mutants, while dlp1 mutants phenocopied the Mpsmxl mutants. Mutations in the KL signaling genes largely suppressed these phenotypes, indicating that DLP1 acts upstream of the KL signaling pathway, although DLP1 also has KL pathway-independent functions. DLP1 exhibited enzymatic activity toward a potential substrate, suggesting the possibility that DLP1 works through KL inactivation. Investigation of DLP1 homologs in A. thaliana revealed that they do not play a major role in the KL pathway, suggesting different mechanisms for the KL signal regulation. Our findings provide new insights into the regulation of the KL signal in M. polymorpha and the evolution of the KL pathway in land plants.

A Polyphenol Oxidase Catalyzes Aurone Synthesis in Marchantia polymorpha.

Aurones constitute one of the major classes of flavonoids, with a characteristic furanone structure that acts as the C-ring of flavonoids. Members of various enzyme families are involved in aurone biosynthesis in different higher plants, suggesting that during evolution plants acquired the ability to biosynthesize aurones independently and convergently. Bryophytes also produce aurones, but the biosynthetic pathways and enzymes involved have not been determined. The present study describes the identification and characterization of a polyphenol oxidase (PPO) that acts as an aureusidin synthase (MpAS1) in the model liverwort, Marchantia polymorpha. Crude enzyme assays using an M. polymorpha line overexpressing MpMYB14 with high accumulation of aureusidin showed that aureusidin was biosynthesized from naringenin chalcone and converted to riccionidin A. This activity was inhibited by N-phenylthiourea, an inhibitor specific to enzymes of the PPO family. Of the six PPOs highly induced in the line overexpressing MpMyb14, one, MpAS1, was found to biosynthesize aureusidin from naringenin chalcone when expressed in Saccharomyces cerevisiae. MpAS1 also recognized eriodictyol chalcone, isoliquiritigenin and butein, showing the highest activity for eriodictyol chalcone. Members of the PPO family in M. polymorpha evolved independently from PPOs in higher plants, indicating that aureusidin synthases evolved in parallel in land plants.

Solanoeclepin B, a hatching factor for potato cyst nematode.

The potato cyst nematode (PCN) causes extensive crop losses worldwide. Because the hatching of PCN requires host-derived molecules known as hatching factors (HFs), regulating HF production in host plants may help to control this harmful pest. Solanoeclepin A (SEA), isolated from potato, is the most active HF for PCN; however, its biosynthesis is completely unknown. We discovered a HF called solanoeclepin B (SEB) from potato and tomato root exudates and showed that SEB was biosynthesized in the plant and converted to SEA outside the plant by biotic agents. Moreover, we identified five SEB biosynthetic genes encoding three 2-oxoglutarate-dependent dioxygenases and two cytochrome P450 monooxygenases in tomato. Exudates from tomato hairy roots in which each of the genes was disrupted contained no SEB and had low hatch-stimulating activity for PCN. These findings will help to breed crops with a lower risk of PCN infection.

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception.

Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl ß-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl ß-D-glucopyranoside arabinosylation to produce HexVic in planta.

Biosynthesis of dillapiole/apiole in dill (Anethum graveolens): characterization of regioselective phenylpropene O-methyltransferase.

The phenylpropene volatiles dillapiole and apiole impart one of the characteristic aromas of dill (Anethum graveolens) weeds. However, very few studies have been conducted to investigate the chemical composition of volatile compounds from different developmental stages and plant parts of A. graveolens. In this study, we examined the distribution of volatile phenylpropenes, including dillapiole, in dill plants at various developmental stages. We observed that young dill seedlings accumulate high levels of dillapiole and apiole, whereas a negligible proportion was found in the flowering plants and dry seeds. Based on transcriptomics and co-expression approaches with phenylpropene biosynthesis genes, we identified dill cDNA encoding S-adenosyl-L-methionine-dependent O-methyltransferase 1 (AgOMT1), an enzyme that can convert 6- and 2-hydroxymyristicin to dillapiole and apiole, respectively, via the methylation of the ortho-hydroxy group. The AgOMT1 protein shows an apparent Km value of 3.5 µm for 6-hydroxymyristicin and is 75% identical to the anise (Pimpinella anisum) O-methyltransferase (PaAIMT1) that can convert isoeugenol to methylisoeugenol via methylation of the hydroxy group at the para-position of the benzene ring. AgOMT1 showed a preference for 6-hydroxymyristicin, whereas PaAIMT1 displayed a large preference for isoeugenol. In vitro mutagenesis experiments demonstrated that substituting only a few residues can substantially affect the substrate specificity of these enzymes. Other plants belonging to the Apiaceae family contained homologous O-methyltransferase (OMT) proteins highly similar to AgOMT1, converting 6-hydroxymyristicin to dillapiole. Our results indicate that apiaceous phenylpropene OMTs with ortho-methylating activity evolved independently of phenylpropene OMTs of other plants and the enzymatic function of AgOMT1 and PaAIMT1 diverged recently.

Tandem Gene Duplication of Dioxygenases Drives the Structural Diversity of Steroidal Glycoalkaloids in the Tomato Clade.

Cultivated tomato (Solanum lycopersicum) contains α-tomatine, a steroidal glycoalkaloid (SGA), which functions as a defense compound to protect against pathogens and herbivores; interestingly, wild species in the tomato clade biosynthesize a variety of SGAs. In cultivated tomato, the metabolic detoxification of α-tomatine during tomato fruit ripening is an important trait that aided in its domestication, and two distinct 2-oxoglutarate-dependent dioxygenases (DOXs), a C-23 hydroxylase of α-tomatine (Sl23DOX) and a C-27 hydroxylase of lycoperoside C (Sl27DOX), are key to this process. There are tandemly duplicated DOX genes on tomato chromosome 1, with high levels of similarity to Sl23DOX. While these DOX genes are rarely expressed in cultivated tomato tissues, the recombinant enzymes of Solyc01g006580 and Solyc01g006610 metabolized α-tomatine to habrochaitoside A and (20R)-20-hydroxytomatine and were therefore named as habrochaitoside A synthase (HAS) and α-tomatine 20-hydroxylase (20DOX), respectively. Furthermore, 20DOX and HAS exist in the genome of wild tomato S. habrochaites accession LA1777, which accumulates habrochaitoside A in its fruits, and their expression patterns were in agreement with the SGA profiles in LA1777. These results indicate that the functional divergence of α-tomatine-metabolizing DOX enzymes results from gene duplication and the neofunctionalization of catalytic activity and gene expression, and this contributes to the structural diversity of SGAs in the tomato clade.

Synthesis of deuterium-labeled cinnamic acids: Understanding the volatile benzenoid pathway in the flowers of the Japanese loquat Eriobotrya japonica.

Cinnamic acids are widely distributed in plants, including crops for human use, and exhibit a variety of activities that are beneficial to human health. They also occupy a pivotal position in the biosynthesis of phenylpropanoids such as lignins, anthocyanins, flavonoids, and coumarins. In this context, deuterium-labeled cinnamic acids have been used as tracers and internal standards in food and medicinal chemistry as well as plant biochemistry. Therefore, a concise synthesis of deuterium-labeled cinnamic acids would be highly desirable. In this study, we synthesized deuterium-labeled cinnamic acids using readily available deuterium sources. We also investigated a hydrogen-deuterium exchange reaction in an ethanol-d1 /Et3 N system. This method can introduce deuterium atoms at the ortho and para positions of the phenolic hydroxy groups as well as at the C-2 position of alkyl cinnamates and is applicable to various phenolic compounds. Using the synthesized labeled compounds, we demonstrated that the benzenoid volatiles, such as 4-methoxybenzaldehyde, in the scent of the flowers of the Japanese loquat Eriobotrya japonica are biosynthesized from phenylalanine via cinnamic and 4-coumaric acids. This study provides easy access to a variety of deuterium-labeled (poly)phenols, as well as to useful tools for studies of the metabolism of cinnamic acids in living systems.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI 498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI 498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

The biosynthetic pathway of potato solanidanes diverged from that of spirosolanes due to evolution of a dioxygenase.

Potato (Solanum tuberosum), a worldwide major food crop, produces the toxic, bitter tasting solanidane glycoalkaloids α-solanine and α-chaconine. Controlling levels of glycoalkaloids is an important focus on potato breeding. Tomato (Solanum lycopersicum) contains a bitter spirosolane glycoalkaloid, α-tomatine. These glycoalkaloids are biosynthesized from cholesterol via a partly common pathway, although the mechanisms giving rise to the structural differences between solanidane and spirosolane remained elusive. Here we identify a 2-oxoglutarate dependent dioxygenase, designated as DPS (Dioxygenase for Potato Solanidane synthesis), that is a key enzyme for solanidane glycoalkaloid biosynthesis in potato. DPS catalyzes the ring-rearrangement from spirosolane to solanidane via C-16 hydroxylation. Evolutionary divergence of spirosolane-metabolizing dioxygenases contributes to the emergence of toxic solanidane glycoalkaloids in potato and the chemical diversity in Solanaceae.

Two BAHD Acyltransferases Catalyze the Last Step in the Shikonin/Alkannin Biosynthetic Pathway.

Several Boraginaceae plants produce biologically active red naphthoquinone pigments, derivatives of the enantiomers shikonin and alkannin, which vary in acyl groups on their side chains. Compositions of shikonin/alkannin derivatives vary in plant species, but the mechanisms generating the diversity of shikonin/alkannin derivatives are largely unknown. This study describes the identification and characterization of two BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1), from Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae that primarily produces the shikonin/alkannin derivatives acetylshikonin and ß-hydroxyisovalerylshikonin. Enzyme assays using Escherichia coli showed that the acylation activity of LeSAT1 was specific to shikonin, whereas the acylation activity of LeAAT1 was specific to alkannin. Both enzymes recognized acetyl-CoA, isobutyryl-CoA, and isovaleryl-CoA as acyl donors to produce their corresponding shikonin/alkannin derivatives, with both enzymes showing the highest activity for acetyl-CoA. These findings were consistent with the composition of shikonin/alkannin derivatives in intact L erythrorhizon plants and cell cultures. Genes encoding both enzymes were preferentially expressed in the roots and cell cultures in the dark in pigment production medium M9, conditions associated with shikonin/alkannin production. These results indicated that LeSAT1 and LeAAT1 are enantiomer-specific acyltransferases that generate various shikonin/alkannin derivatives.

Effects of a pyroglutamyl pentapeptide isolated from fermented barley extract on atopic dermatitis-like skin lesions in hairless mouse.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic and eczematous skin lesions. The skin of AD patients is generally in a dried condition. Therefore, it is important for AD patients to manage skin moisturization. In this study, we examined the effects of orally administered fermented barley extract P (FBEP), which is prepared from a supernatant of barley shochu distillery by-product, on stratum corneum (SC) hydration and transepidermal water loss (TEWL) in AD-like lesions induced in hairless mice using 2,4,6-trinitrochlorobenzene. Oral administration of FBEP increased SC hydration and decreased TEWL in the dorsal skin of this mouse model. Further fractionation of FBEP showed that a pyroglutamyl pentapeptide, pEQPFP comprising all -L-form amino acids, is responsible for these activities. These results suggested that this pyroglutamyl pentapeptide may serve as a modality for the treatment of AD.

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity.

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity. ABBREVIATIONS: ACP: acyl carrier protein; CAPS: N-cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-ß-D-maltoside; DDM: n-dodecyl-ß-D-maltoside; DTNB: 5,5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid.

Identification of α-Tomatine 23-Hydroxylase Involved in the Detoxification of a Bitter Glycoalkaloid.

Tomato plants (Solanum lycopersicum) contain steroidal glycoalkaloid α-tomatine, which functions as a chemical barrier to pathogens and predators. α-Tomatine accumulates in all tissues and at particularly high levels in leaves and immature green fruits. The compound is toxic and causes a bitter taste, but its presence decreases through metabolic conversion to nontoxic esculeoside A during fruit ripening. This study identifies the gene encoding a 23-hydroxylase of α-tomatine, which is a key to this process. Some 2-oxoglutarate-dependent dioxygenases were selected as candidates for the metabolic enzyme, and Solyc02g062460, designated Sl23DOX, was found to encode α-tomatine 23-hydroxylase. Biochemical analysis of the recombinant Sl23DOX protein demonstrated that it catalyzes the 23-hydroxylation of α-tomatine and the product spontaneously isomerizes to neorickiioside B, which is an intermediate in α-tomatine metabolism that appears during ripening. Leaves of transgenic tomato plants overexpressing Sl23DOX accumulated not only neorickiioside B but also another intermediate, lycoperoside C (23-O-acetylated neorickiioside B). Furthermore, the ripe fruits of Sl23DOX-silenced transgenic tomato plants contained lower levels of esculeoside A but substantially accumulated α-tomatine. Thus, Sl23DOX functions as α-tomatine 23-hydroxylase during the metabolic processing of toxic α-tomatine in tomato fruit ripening and is a key enzyme in the domestication of cultivated tomatoes.

Involvement of γ-Glutamyl Transpeptidase in Ischemia/Reperfusion-Induced Cardiac Dysfunction in Isolated Rat Hearts.

GGsTop is a highly potent and specific, and irreversible γ-glutamyl transpeptidase (GGT) inhibitor without any influence on glutamine amidotransferases. The aim of the present study was to investigate the involvement of GGT in ischemia/reperfusion-induced cardiac dysfunction by assessing the effects of a treatment with GGsTop. Using a Langendorff apparatus, excised rat hearts underwent 40 min of global ischemia without irrigation and then 30 min of reperfusion. GGT activity was markedly increased in cardiac tissues exposed to ischemia, and was inhibited by the treatment with GGsTop. Exacerbation of cardiac functional parameters caused by ischemia and reperfusion, namely the reduction of left ventricular (LV) developed pressure and the maximum and negative minimum values of the first derivative of LV pressure, and the increment in LV end-diastolic pressure was significantly attenuated by GGsTop treatment. The treatment with GGsTop suppressed excessive norepinephrine release in the coronary perfusate, a marker for myocardial dysfunction, after ischemia/reperfusion. In addition, oxidative stress indicators in myocardium, including superoxide and malondialdehyde, after ischemia/reperfusion were significantly low in the presence of GGsTop. These observations demonstrate that enhanced GGT activity contributes to cardiac damage after myocardial ischemia/reperfusion, possibly via increased oxidative stress and subsequent norepinephrine overflow. GGT inhibitors have potential as a therapeutic strategy to prevent myocardial ischemia/reperfusion injury in vivo.

Characterization of melanin and optimal conditions for pigment production by an endophytic fungus, Spissiomyces endophytica SDBR-CMU319.

Melanin is a natural pigment that is produced by filamentous fungi. In this study, the endophytic species, Spissiomyces endophytica (strain SDBR-CMU319), produced a brown-black pigment in the mycelia. Consequently, the pigment was extracted from the dried fungal biomass. This was followed by pigment purification, characterization and identification. Physical and chemical characteristics of the pigment showed acid precipitation, alkali solubilization, decolorization with oxidizing agents, and insolubility in most organic solvents and water. The pigment was confirmed as melanin based on ultraviolet-visible spectroscopy, Fourier-transform infrared, and electron paramagnetic resonance spectra analyses. The analyses of the elemental composition indicated that the pigment possessed a low percentage of nitrogen, and therefore, was not 3,4-dihydroxyphenylalanine melanin. Inhibition studies involving specific inhibitors, both tricyclazole and phthalide, and suggest that fungal melanin could be synthesized through the 1,8-dihydroxynaphthalene pathway. The optimum conditions for fungal pigment production from this species were investigated. The highest fungal pigment yield was observed in glucose yeast extract peptone medium at an initial pH value of 6.0 and at 25°C over three weeks of cultivation. This is the first report on the production and characterization of melanin obtained from the genus Spissiomyces.

Structural insights into a key step of brassinosteroid biosynthesis and its inhibition.

Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development1. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs2. Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor3-5. We also determined the crystal structures of CYP90B1 complexed with uniconazole6,7 or brassinazole8, which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.