Hordeum vulgare CYP76M57 catalyzes C2 shortening of tryptophan side chain by C-N bond rearrangement in gramine biosynthesis.

The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C2 shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and Cα and the rearrangement of the nitrogen atom to Cß. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.

Detection of a novel intramolecular rearrangement during gramine biosynthesis in barley using stable isotope-labeled tryptophan.

Plants accumulate various secondary metabolites, and the biosynthetic reactions responsible for their scaffold construction are the key steps that characterize their structural categories. Gramine, an indole alkaloid, is a defensive secondary metabolite biosynthesized in barley (Hordeum vulgare) from tryptophan (Trp) via aminomethylindole (AMI). While the two sequential N-methylation steps following the formation of AMI have already been characterized both genetically and enzymatically, the step preceding AMI formation, which includes the Trp side chain-shortening, has not yet been revealed. To gain further insight into these biosynthetic reactions, barley seedlings were fed Trp labeled with stable isotopes (13C and 15N) at various positions, and the isotope incorporation into gramine was analyzed by liquid chromatography/mass spectrometry. Significant increases in the abundance of isotopic gramine were detected in experimental sets in which Trp was labeled at either the indole ring, the ß-carbon, or the amino group, whereas the isotopolog composition was not affected by α-carbon-labeled Trp. Although absorbed Trp presumably undergoes transamination in plants, this reaction did not seem to be related to gramine productivity. The data indicated that AMI directly inherited the amino group from Trp, while the α-carbon was removed, suggesting that the Trp-AMI conversion includes a novel intramolecular rearrangement reaction. The results of this study provide novel insights into scaffold formation in plant secondary-metabolite synthesis.

Isolation of phenolic acids and tannin acids from Mangifera indica L. kernels as inhibitors of lipid accumulation in 3T3-L1 cells.

Mango (Mangifera indica L.) kernels are usually discarded as waste, but they contain many pharmacological properties and bioactivities. In this study, we isolated antiobesity agents from mango kernels that inhibit intracellular lipid formation in 3T3-L1 adipocytes. Two phenolic acids, ethyl gallate and ethyl digallate, and 2 tannin acids, 1,2,3,4,6-penta-O-galloyl-ß-d-glucose (PGG) and 3-O-digalloyl-1,2,4,6-tetra-O-ß-d-glucose (HGG), were identified from mango kernels and were found to be suppressed lipid accumulation as evidenced by Oil Red O staining. Furthermore, ethyl digallate, PGG, and HGG significantly downregulated the mRNA expression of adipogenic transcription factors such as C/EBPα and PPARγ. However, ethyl gallate did not affect the expression of these transcription factors. Our findings reveal the presence of antiobesity compounds in mango kernels, implying its therapeutic role against obesity.

Increased benzoxazinoid (Bx) levels in wheat seedlings via jasmonic acid treatment and etiolation and their effects on Bx genes including Bx6.

Wheat accumulates benzoxazinoid (Bx) as a defensive compound. While Bx occurs at high concentrations, particularly in the early growth stages, its mechanism of regulation remains unclear. In the present study, we first examined the effects of several plant hormones on Bx concentrations in wheat seedlings. Among the compounds tested, jasmonate (JA) elevated the concentrations of DIMBOA-Glc (2-ß-D-glucoside of 2,4-dihydroxy-7-methoy-1,4-benzoxazin-3-one), the primary Bx species in intact wheat seedlings, without a significant increase in HDMBOA-Glc (4-O-methyl-DIMBOA-Glc), which is known to be upregulated by stresses. In addition, growing the plants in the dark increased DIMBOA-Glc levels. Quantification of the Bx-biosynthetic genes showed that TaBx8 (UDP-Glc:Bx glucosyltrasferase) was influenced by neither JA nor etiolation, indicating that TaBx8 is under the regulation mechanism distinct from the mechanisms influencing the others. In addition, none of the other gene expression patterns exhibited considerable correlation with DIMBOA-Glc accumulation. Since there was no correlation between transcript levels of the genes involved in Bx biosynthesis and Bx accumulation, other factors may control the levels of Bx in wheat. In the course of gene analyses, we isolated TaBx6, one of the last two genes that had not been identified in wheat in the DIMBOA-Glc biosynthetic pathway. All the four TaBx6 genes cloned in the present study were expressed in Escherichia coli and characterized their activity.

Molecular and structural characterization of agmatine coumaroyltransferase in Triticeae, the key regulator of hydroxycinnamic acid amide accumulation.

Hydroxycinnamic acid amides (HCAAs) are involved in stress-induced defense in many plant species. Barley accumulates high concentrations of HCAAs irrespective of exogenous stressors, while other major cereals such as wheat and rice accumulate relatively low levels of HCAAs in intact tissues. The primary HCAA species in barley are biosynthesized by agmatine p-coumaroyltransferase (ACT), an N-acyltransferase of the BAHD superfamily. However, the molecular basis underlying barley's uniquely high HCAA accumulation has not been elucidated, and information regarding the structural details of BAHD N-acyltransferases is limited. Hence, we aimed to investigate the ACTs of family Poaceae. We isolated ACT (-like) genes, including those previously undescribed, and investigated their enzymatic and genetic features. All the identified enzymes belonged to clade IVa of the BAHD superfamily. The barley and wheat ACTs were further categorized, based on catalytic properties and primary structures, into ACT1 and ACT2 groups, the encoding loci of which are neighbors on the same chromosome. While all ACTs exhibited similar Km values for CoA-thioesters (acyl-group donors), members of the ACT1 group showed a distinctly higher affinity for agmatine (acyl-acceptor). Among the ACTs tested, an ACT isozyme in barley (HvACT1-1) showed the highest catalytic efficiency and transcript level, indicating that ACT regulates high-level HCAA accumulation in barley. For further enzymatic characterization of the ACTs, we crystalized wheat ACT2 (TaACT2) and determined its structure at 2.3 Å resolution. Structural alignment of TaACT2 and HvACT1-1 showed that the architectures of the substrate binding pockets were well conserved. However, the structure of a loop located at the entrance to acyl-acceptor binding site may be more flexible in TaACT2, which could be responsible for the lower affinity of TaACT2 to agmatine. Mutations of HvACT1-1 at Glu372 and Asp374 within one of the clade-IV specific motifs facing the deduced acyl-acceptor binding pocket caused significant catalytic deterioration toward agmatine both in Km and kcat, suggesting their key roles in acyl acceptor binding by the clade-IV enzymes. This study elucidated the molecular basis of how plants accumulate defensive specialized metabolites and provided insights into developing efficient and eco-friendly agricultural methods.

Identification of methoxylchalcones produced in response to CuCl2 treatment and pathogen infection in barley.

Changes in specialized metabolites were analyzed in barley (Hordeum vulgare) leaves treated with CuCl2 solution as an elicitor. LC-MS analysis of the CuCl2-treated leaves showed the induced accumulation of three compounds. Among them, two were purified by silica gel and ODS column chromatography and preparative HPLC and were identified as 2',3,4,4',6'-pentamethoxychalcone and 2'-hydroxy-3,4,4',6'-tetramethoxychalcone by spectroscopic analyses. The remaining compound was determined as 12-oxo-phytodienoic acid (OPDA), a major oxylipin in plants, by comparing its spectrum and retention time from LC-MS/MS analysis with those of the authentic compound. The accumulation of these compounds was reproduced in leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of the Poaceae species. This inoculation increased the amounts of other oxylipins, including jasmonic acid (JA), JA-Ile, 9-oxooctadeca-10,12-dienoic acid (9-KODE), and 13-oxooctadeca-9,11-dienoic acid (13-KODE). The treatments of the barley leaves with JA and OPDA induced the accumulation of methoxylchalcones, but treatment with 9-KODE did not. These methoxylchalcones inhibited conidial germination of B. sorokiniana and Fusarium graminearum, thereby indicating that these compounds possessed antifungal activity. Consequently, they are considered to be involved in the chemical defense processes as phytoalexins in barley. Accumulation of methoxylchalcones in response to JA treatment was observed in all seven barley cultivars tested, but was not detected in other wild Hordeum species, wheat, and rice, thus indicating that their production was specific to cultivated barley.

Identification of an antiviral component from the venom of the scorpion Liocheles australasiae using transcriptomic and mass spectrometric analyses.

Scorpion venom contains a variety of biologically active peptides. Among them, neurotoxins are major components in the venom, but it also contains peptides that show antimicrobial activity. Previously, we identified three insecticidal peptides from the venom of the Liocheles australasiae scorpion, but activities and structures of other venom components remained unknown. In this study, we performed a transcriptome analysis of the venom gland of the scorpion L. australasiae to gain a comprehensive understanding of its venom components. The result shows that potassium channel toxin-like peptides were the most diverse, whereas only a limited number of sodium channel toxin-like peptides were observed. In addition to these neurotoxin-like peptides, many non-disulfide-bridged peptides were identified, suggesting that these components have some critical roles in the L. australasiae venom. In this study, we also isolated a component with antiviral activity against hepatitis C virus using a bioassay-guided fractionation approach. By integrating mass spectrometric and transcriptomic data, we successfully identified LaPLA2-1 as an anti-HCV component. LaPLA2-1 is a phospholipase A2 having a heterodimeric structure that is N-glycosylated at the N-terminal region. Since the antiviral activity of LaPLA2-1 was inhibited by a PLA2 inhibitor, the enzymatic activity of LaPLA2-1 is likely to be involved in its antiviral activity.

Crystal structure of barley agmatine coumaroyltransferase, an N-acyltransferase from the BAHD superfamily.

The enzymes of the BAHD superfamily, a large group of acyl-CoA-dependent acyltransferases in plants, are involved in the biosynthesis of diverse secondary metabolites. While the structures of several O-acyltransferases from the BAHD superfamily, such as hydroxycinnamoyl-CoA shikimate hydroxycinnamoyl transferase, have been elucidated, no structural information on N-acyltransferases is available. Hordeum vulgare agmatine coumaroyltransferase (HvACT) is an N-acyltransferase from the BAHD superfamily and is one of the most important enzymes in the secondary metabolism of barley. Here, an apo-form structure of HvACT is reported as the first structure of an N-acyltransferase from the BAHD superfamily. HvACT crystals diffracted to 1.8 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 57.6, b = 59.5, c = 73.6 Å, α = 90, ß = 91.3 , γ = 90°. Like other known BAHD superfamily structures, HvACT contains two domains that adopt a two-layer αß-sandwich architecture and a solvent-exposed channel that penetrates the enzyme core.

Crystal structure of the delta-class glutathione transferase in Musca domestica.

Among the various glutathione transferase (GST) isozymes in insects, the delta- and epsilon-class GSTs fulfill critical functions during the detoxification of insecticides. We crystalized MdGSTD1, the major delta-class GST isozyme in the housefly (Musca domestica), in complex with glutathione (GSH) and solved its structure at a resolution of 1.4 Å. The overall folding of MdGSTD1 resembled other known delta-class GSTs. Its substrate binding pocket was exposed to solvent and considerably more open than in the epsilon-class GST from M. domestica (MdGSTE2). However, their C-terminal structures differed the most because of the different lengths of the C-terminal regions. Although this region does not seem to directly interact with substrates, its deletion reduced the enzymatic activity by more than 70%, indicating a function in maintaining the proper conformation of the binding pocket. Binding of GSH to the GSH-binding region of MdGSTD1 results in a rigid conformation of this region. Although MdGSTD1 has a higher affinity for GSH than the epsilon class enzymes, the thiol group of the GSH molecule was not close enough to serine residue 9 to form a hydrogen-bond with this residue, which is predicted to act as the catalytic center for thiol group deprotonation in GSH.

Structure-Based Chemical Design of Abscisic Acid Antagonists That Block PYL-PP2C Receptor Interactions.

In Arabidopsis, signaling of the stress hormone abscisic acid (ABA) is mediated by PYR/PYL/RCAR receptors (PYLs), which bind to and inhibit group-A protein phosphatases 2C (PP2Cs), the negative regulators of ABA. X-ray structures of several PYL-ABA and PYL-ABA-PP2C complexes have revealed that a conserved tryptophan in PP2Cs is inserted into a small tunnel adjacent to the C4' of ABA in the PYL-ABA complex and plays a crucial role in the formation and stabilization of the PYL-ABA-PP2C complex. Here, 4'-modified ABA analogues were designed to prevent the insertion of the tryptophan into the tunnel adjacent to the C4' of ABA in these complexes. These analogues were predicted to block PYL-PP2C receptor interactions and thus block ABA signaling. To test this, 4'- O-phenylpropynyl ABA analogues were synthesized as novel PYL antagonists (PANs). Structural, thermodynamic, biochemical, and physiological studies demonstrated that PANs completely abolished ABA-induced PYL-PP2C interactions in vitro and suppressed stress-induced ABA responses in vivo more strongly than did 3'-hexylsulfanyl-ABA (AS6), a PYL antagonist we developed previously. The PANs and AS6 antagonized the effects of ABA to different degrees in different plants, suggesting that these PANs can function as chemical scalpels to dissect the complicated regulatory mechanism of ABA signaling in plants.

Detoxification process of tolaasins, lipodepsipeptides, by Microbacterium sp. K3-5.

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.

Elongation of barley roots in high-pH nutrient solution is supported by both cell proliferation and differentiation in the root apex.

Many crops grow well on neutral or weakly acidic soils. The ability of roots to elongate under high-external pH would be advantageous for the survival of plants on alkaline soil. We found that root elongation was promoted in some plant species in alkaline-nutrient solution. Barley, but not tomato, root growth was maintained in pH 8 nutrient solution. Fe and Mn were absorbed well from the pH 8 nutrient solution by both barley and tomato plants, suggesting that the different growth responses of these two species may not be caused by insolubilization of transition metals. The ability of intact barley and tomato plants to acidify external solution was comparable; in both species, this ability decreased in plants exposed to pH 8 nutrient solution for 1 w. Conversely, cell proliferation and elongation in barley root apices were facilitated at pH 8 as shown by microscopy and cell-cycle-related gene-expression data; this was not observed in tomato. We propose that barley adapts to alkaline stress by increasing root development.

Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

Structural analysis of an epsilon-class glutathione transferase from housefly, Musca domestica.

Glutathione transferases (GSTs) play an important role in the detoxification of insecticides, and as such, they are a key contributor to enhanced resistance to insecticides. In the housefly (Musca domestica), two epsilon-class GSTs (MdGST6A and MdGST6B) that share high sequence homology have been identified, which are believed to be involved in resistance against insecticides. The structural determinants controlling the substrate specificity and enzyme activity of MdGST6s are unknown. The aim of this study was to crystallize and perform structural analysis of the GST isozyme, MdGST6B. The crystal structure of MdGST6B complexed with reduced glutathione (GSH) was determined at a resolution of 1.8 Å. MdGST6B was found to have a typical GST folding comprised of N-terminal and C-terminal domains. Arg113 and Phe121 on helix 4 were shown to protrude into the substrate binding pocket, and as a result, the entrance of the substrate binding pocket was narrower compared to delta- and epsilon-class GSTs from Africa malaria vector Anopheles gambiae, agGSTd1-6 and agGSTe2, respectively. This substrate pocket narrowing is partly due to the presence of a π-helix in the middle of helix 4. Among the six residues that donate hydrogen bonds to GSH, only Arg113 was located in the C-terminal domain. Ala substitution of Arg113 did not have a significant effect on enzyme activity, suggesting that the Arg113 hydrogen bond does not play a crucial role in catalysis. On the other hand, mutation at Phe108, located just below Arg113 in the binding pocket, reduced the affinity and catalytic activity to both GSH and the electrophilic co-substrate, 1-chloro-2,4-dinitrobenzene.

Dispersed benzoxazinone gene cluster: molecular characterization and chromosomal localization of glucosyltransferase and glucosidase genes in wheat and rye.

Benzoxazinones (Bxs) are major defensive secondary metabolites in wheat (Triticum aestivum), rye (Secale cereale), and maize (Zea mays). Here, we identified full sets of homeologous and paralogous genes encoding Bx glucosyltransferase (GT) and Bx-glucoside glucosidase (Glu) in hexaploid wheat (2n = 6x = 42; AABBDD). Four GT loci (TaGTa-TaGTd) were mapped on chromosomes 7A, 7B (two loci), and 7D, whereas four glu1 loci (Taglu1a-Taglu1d) were on chromosomes 2A, 2B (two loci), and 2D. Transcript levels differed greatly among the four loci; B-genome loci of both TaGT and Taglu1 genes were preferentially transcribed. Catalytic properties of the enzyme encoded by each homeolog/paralog also differed despite high levels of identity among amino acid sequences. The predominant contribution of the B genome to GT and Glu reactions was revealed, as observed previously for the five Bx biosynthetic genes, TaBx1 to TaBx5, which are separately located on homeologous groups 4 and 5 chromosomes. In rye, where the ScBx1 to ScBx5 genes are dispersed to chromosomes 7R and 5R, ScGT and Scglu were located separately on chromosomes 4R and 2R, respectively. The dispersal of Bx-pathway loci to four distinct chromosomes in hexaploid wheat and rye suggests that the clustering of Bx-pathway genes, as found in maize, is not essential for coordinated transcription. On the other hand, barley (Hordeum vulgare) was found to lack the orthologous GT and glu loci like the Bx1 to Bx5 loci despite its close phylogenetic relationship with wheat and rye. These results contribute to our understanding of the evolutionary processes that the Bx-pathway loci have undergone in grasses.

Active-site architecture of benzoxazinone-glucoside ß-D-glucosidases in Triticeae.

The ß-D-glucosidases from wheat (Triticum aestivum) and rye (Secale cereale) hydrolyze benzoxazinone-glucose conjugates. Although wheat and rye glucosidases have high sequence identity, they have different substrate preferences; the wheat enzyme favors DIMBOA-Glc (2-O-ß-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-Glc (7-demethoxy-DIMBOA-Glc), whereas the rye enzyme preference is the opposite. To investigate the mechanism of substrate binding, we analyzed crystal structures of an inactive mutant of the wheat glucosidase complexed with the natural substrate DIMBOA-Glc, wheat and rye glucosidases complexed with an aglycone DIMBOA, and wheat and rye glucosidases complexed with an inhibitor 2-fluoro-2-deoxy-ß-D-glucose. The binding position of substrate in the active site was determined but interaction between the substrate and Ser-464 or Leu-465 was not observed, although amino acid residues at these two positions are the only structural distinctions between wheat and rye glucosidase catalytic pockets. Variation at these two positions alters the width of the pocket entrance, which may relate to observed differences in substrate specificity. The side chain of Glu-462 that forms hydrogen bonds with the glucose moiety of DIMBOA-Glc moved deeper into the pocket upon substrate binding, and mutation of this residue dramatically decreased enzyme activity.

Isolation and structure elucidation of tumescenamides A and B, two peptides produced by Streptomyces tumescens YM23-260.

Two peptides, tumescenamides A and B, were isolated from the fermentation broth of a marine bacterium, Streptomyces tumescens YM23-260. The structure of tumescenamide A was determined to be a cyclic depsipeptide consisting of α-amino-2-butenoic acid, tyrosine, valine, leucine and threonine, substituted with a 2,4-dimethylheptanoyl residue at the α-NH(2) position. Tumescenamide B possesses a 2,4,6-trimethylnonanoyl residue in place of the 2,4-dimethylheptanoyl substituent in tumescenamide A. Tumescenamide A induced reporter gene expression under the control of the insulin-degrading enzyme promoter.

Studies on terpenoids produced by actinomycetes. 5-dimethylallylindole-3-carboxylic Acid and A80915G-8"-acid produced by marine-derived Streptomyces sp. MS239.

As a result of screening for terpenoids produced by marine-derived Streptomyces sp. MS239, two new terpenoids named 5-dimethylallylindole-3-carboxylic acid and A80915G-8''-acid were isolated and their structures were determined mainly by NMR analyses.

Terpenoids Produced by Actinomycetes: Napyradiomycins from Streptomyces antimycoticus NT17.

Napyradiomycin SR ( 1), 16-dechloro-16-hydroxynapyradiomycin C2 ( 2), 18-hydroxynapyradiomycin A1 ( 3), 18-oxonapyradiomycin A1 ( 4), 16-oxonapyradiomycin A2 ( 5), 7-demethyl SF2415A3 ( 6), 7-demethyl A80915B ( 7), and ( R)-3-chloro-6-hydroxy-8-methoxy-alpha-lapachone ( 8) were isolated from the culture broth of Streptomyces antimycoticus NT17. These compounds are derivatives of the napyradiomycins isolated previously from Chainia rubra or Streptomyces aculeolatus. The structures of the new compounds, some of which exhibit antibacterial activities, were established by comparing their NMR data with data of related known compounds. The unique structure of 1, containing a highly strained ring, was established by NMR and was confirmed by X-ray analysis. Two of the compounds are C-16 stereoisomers of napyradiomycin A2 and are named napyradiomycins A2a ( 9a) and A2b ( 9b).

Studies on terpenoids produced by actinomycetes: oxaloterpins A, B, C, D, and E, diterpenes from Streptomyces sp. KO-3988.

Following screening for terpenoids produced by Streptomyces sp. KO-3988, five new diterpenes named oxaloterpins A (1), B (2), C (3), D (4), and E (5) together with the known viguiepinone (6) were isolated from culture broth, and their structures were established on the basis of extensive NMR and MS analyses. The absolute configuration of oxaloterpin A was determined by the modified Mosher's method as 3 R, 5 S, 8 S, 10 R, 13 S.