Structural insights into catalytic promiscuity of chalcone synthase from Glycine max (L.) Merr.: Coenzyme A-induced alteration of product specificity.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC50, 67 µM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production.

Fermented food Tempeh induces interleukin 12 and enhances macrophage phagocytosis.

It is known that lactic acid bacteria induce the IL-12. The IL-12 activates NK cells and promotes the production of IFN-γ. The IFN-γ activates macrophages resulting in enhanced phagocytosis and bactericidal activity. We have been investigating fermented foods that activate the immune function. In this study, we investigated the IL-12 inducibility of fermented foods using the specific antibody. Fermented soybean foods such as Tempeh and Natto are attracting attention in terms of nutrition, functionality, and food problems. In this study, Tempeh induced 1,080 µg/ml of IL-12, and IFN-γ associated with the induction of IL-12 was also induced at 682 µg/ml. This was more than twice the induced intensity of PBS. On the contrary, Natto hardly induced IL-12 and IFN-γ. Tempeh also accelerated phagocytosis of the macrophage THP-1 cells. In this study, it was found that the fermented soybean-derived food, Tempeh, has a function of activating the immune function. This is the first report that Tempeh activates innate immunity. PRACTICAL APPLICATIONS: Tempeh, a fermented soybean food induced the IL-12 and IFN-γ production and the increase of macrophage phagocytosis in this study suggested a new function to enhance immunity. Tempeh is also expected to be effective in preventing lifestyle diseases. Fermented soybean products of Tempeh was considered to be a very useful health food for the problems of modern society such as maintaining health by eating, improving immunity, and ingesting vegetable protein due to diversifying food.

A conserved strategy of chalcone isomerase-like protein to rectify promiscuous chalcone synthase specificity.

Land plants produce diverse flavonoids for growth, survival, and reproduction. Chalcone synthase is the first committed enzyme of the flavonoid biosynthetic pathway and catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC). However, it also produces other polyketides, including p-coumaroyltriacetic acid lactone (CTAL), because of the derailment of the chalcone-producing pathway. This promiscuity of CHS catalysis adversely affects the efficiency of flavonoid biosynthesis, although it is also believed to have led to the evolution of stilbene synthase and p-coumaroyltriacetic acid synthase. In this study, we establish that chalcone isomerase-like proteins (CHILs), which are encoded by genes that are ubiquitous in land plant genomes, bind to CHS to enhance THC production and decrease CTAL formation, thereby rectifying the promiscuous CHS catalysis. This CHIL function has been confirmed in diverse land plant species, and represents a conserved strategy facilitating the efficient influx of substrates from the phenylpropanoid pathway to the flavonoid pathway.

Microbial Diversity in the Edible Gall on White Bamboo Formed by the Interaction between Ustilago esculenta and Zizania latifolia.

An edible gall is formed between the third and fourth nodes beneath the apical meristem near the base of Zizania latifolia shoots. This gall is harbored by and interacts with the smut fungus Ustilago esculenta. The gall is also a valuable vegetable called "white bamboo," jiaobai or gausun in China and makomotake in Japan. Five samples of the galls harvested at different stages of swelling were used to isolate microorganisms by culturing. Isolated fungal and bacterial colonies were identified by DNA sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, respectively. Several strains of U. esculenta as well as 6 other species of fungi and 10 species of bacteria were isolated. The microbiome was also evaluated by simple and outlined DNA profiling with automated rRNA intergenic spacer analysis (ARISA), and the amount of DNA of U. esculenta was determined by qPCR. At least 16 species of fungi and 40 species of bacteria were confirmed by ARISA of the overall sample. Interestingly, the greatest bacterial diversity, i.e., 18 species, was observed in the most mature sample, whereas the fungal diversity observed in this sample, i.e., 4 species, was rather poor. Based on qPCR, U. esculenta occurred in samples from all stages; however, the abundance of U. esculenta exhibited unique U-shaped relationships with growth. These results may explain why the interaction between U. esculenta and Z. latifolia also influences the unique microbial diversity observed throughout the growth stages of the swollen shoot, although the limited sample size does not allow conclusive findings.

Near-Infrared Emitting PbS Quantum Dots for in Vivo Fluorescence Imaging of the Thrombotic State in Septic Mouse Brain.

Near-infrared (NIR) fluorescent imaging is a powerful tool for the non-invasive visualization of the inner structure of living organisms. Recently, NIR fluorescence imaging at 1000-1400 nm (second optical window) has been shown to offer better spatial resolution compared with conventional NIR fluorescence imaging at 700-900 nm (first optical window). Here we report lead sulfide (PbS) quantum dots (QDs) and their use for in vivo NIR fluorescence imaging of cerebral venous thrombosis in septic mice. Highly fluorescent PbS QDs with a 1100 nm emission peak (QD1100) were prepared from lead acetate and hexamethyldisilathiane, and the surface of QD1100 was coated with mercaptoundecanoic acid so as to be soluble in water. NIR fluorescence imaging of the cerebral vessels of living mice was performed after intravascular injection (200-300 µL) of QD1100 (3 µM) from a caudal vein. By detecting the NIR fluorescence of QD1100, we achieved non-invasive NIR fluorescence imaging of cerebral blood vessels through the scalp and skull. We also achieved NIR fluorescence imaging of cerebral venous thrombosis in septic mice induced by the administration of lipopolysaccharide (LPS). From the NIR fluorescence imaging, we found that the number of thrombi in septic mice was significantly increased by the administration of LPS. The formation of thrombi in cerebral blood vessels in septic mice was confirmed by enzyme-linked immunosorbent assay (ELISA). We also found that the number of thrombi significantly decreased after the administration of heparin, an inhibitor of blood coagulation. These results show that NIR fluorescence imaging with QD1100 is useful for the evaluation of the pathological state of cerebral blood vessels in septic mice.

Rapid and reliable species identification of wild mushrooms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

Mushrooms are a favourite natural food in many countries. However, some wild species cause food poisoning, sometimes lethal, due to misidentification caused by confusing fruiting bodies similar to those of edible species. The morphological inspection of mycelia, spores and fruiting bodies have been traditionally used for the identification of mushrooms. More recently, DNA sequencing analysis has been successfully applied to mushrooms and to many other species. This study focuses on a simpler and more rapid methodology for the identification of wild mushrooms via protein profiling based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). A preliminary study using 6 commercially available cultivated mushrooms suggested that a more reproducible spectrum was obtained from a portion of the cap than from the stem of a fruiting body by the extraction of proteins with a formic acid-acetonitrile mixture (1 + 1). We used 157 wild mushroom-fruiting bodies collected in the centre of Hokkaido from June to November 2014. Sequencing analysis of a portion of the ribosomal RNA gene provided 134 identifications of mushrooms by genus or species, however 23 samples containing 10 unknown species that had lower concordance rate of the nucleotide sequences in a BLAST search (less than 97%) and 13 samples that had unidentifiable poor or mixed sequencing signals remained unknown. MALDI-TOF MS analysis yielded a reproducible spectrum (frequency of matching score ≥ 2.0 was ≥6 spectra from 12 spectra measurements) for 114 of 157 samples. Profiling scores that matched each other within the database gave correct species identification (with scores of ≥2.0) for 110 samples (96%). An in-house prepared database was constructed from 106 independent species, except for overlapping identifications. We used 48 wild mushrooms that were collected in autumn 2015 to validate the in-house database. As a result, 21 mushrooms were identified at the species level with scores ≥2.0 and 5 mushrooms at the genus level with scores ≥1.7, although the signals of 2 mushrooms were insufficient for analysis. The remaining 20 samples were recognized as "unreliable identification" with scores <1.7. Subsequent DNA analysis confirmed that the correct species or genus identifications were achieved by MALDI-TOF MS for the 26 former samples, whereas the 18 mushrooms with poorly matched scores were species that were not included in the database. Thus, the proposed MALDI-TOF MS coupled with our database could be a powerful tool for the rapid and reliable identification of mushrooms; however, continuous updating of the database is necessary to enrich it with more abundant species.

Identification of Mushroom Species by Automated rRNA Intergenic Spacer Analysis (ARISA) and Its Application to a Suspected Case of Food Poisoning with Tricholoma ustale.

Automated rRNA intergenic spacer analysis (ARISA), a method of microbiome analysis, was evaluated for species identification of mushrooms based on the specific fragment sizes. We used 51 wild mushroom-fruiting bodies collected in the centre of Hokkaido and two cultivated mushrooms. Samples were hot-air-dried and DNA were extracted by a beads beating procedure. Sequencing analysis of portions of the rRNA gene (rDNA) provided 33 identifications of mushrooms by genus or species. The results of ARISA identification based on the combination of the fragment sizes corresponding to two inter spacer regions (ITS2 and ITS1) of rDNA within±0.1% accuracy showed that 27 out of the 33 species had specific fragment sizes differentiated from other species. The remaining 6 species formed 3 pairs that showed overlapping fragment sizes. In addition, within-species polymorphisms were observed as 1 bp differences among 32 samples of 13 species. ARISA was applied to investigate a case of suspected food poisoning in which the mushroom was thought to be a toxic Kakishimeji. The morphological identification of the mushroom was ambiguous since the remaining sample lacked a part of the fruiting body. Further, yeast colonies had grown on the surface of the fruiting body during storage. The ARISA fragment size of the mushroom showed 7 bp difference from that of the candidate toxic mushroom. Although ARISA could be a useful tools for estimation of mushroom species, especially in case where the fruiting bodies have deteriorated or been processed, further studies are necessary for reliable identification. For example, it may be necessary to adopt more informative genes which could provide clearer species-specific polymorphisms than the ITS regions.