Reductive dehalogenase (RDase) consists of two parts, RdhA and RdhB. RdhA is the catalytic subunit, harboring a cobalamin cofactor and two Fe-S clusters. RdhA is anchored to the cytoplasmic membrane via the membrane anchoring subunit, RdhB. There are many genes encoding RDases in the genome of organohalide-respiring bacteria, including Dehalococcoides spp. However, most genes have not been functionally characterized. Biochemical studies on RDases have been hampered by difficulties encountered in their expression and purification. In this study, we have expressed, purified and characterized RdhA of RDase for tetrachloroethene (PceA) from Geobacter sp. PceA was expressed as a fusion protein with a trigger factor tag in Escherichia coli. PceA was purified and denatured in aerobic condition. Subsequently, this protein was refolded in the presence of FeCl3, Na2S and cobalamin in anaerobic condition. The reconstituted PceA exhibited dechlorination ability for tetrachloroethene. UV-Vis spectroscopy has shown that it contains cobalamin and Fe-S clusters. Since this method requires anaerobic manipulation only in the reconstituting process and has a relatively high yield, it will enable further biochemical studies of RDases.

Here, we investigated whether the optimal threonine (Thr) to lysine (Lys) ratio in high Lys diet improves the growth performance of modern broiler chickens at finisher period and determined the possible mechanism underlying improvement in the growth performance of chickens fed with high Lys or Lys + Thr diet using metabolome analyses. Eighteen 21-day-old chickens housed in individual cages were randomly divided into three groups of six chickens fed with different diets as follows: control diet, high Lys diet (150% Lys content of National Research Council requirement), and high Lys + Thr diet (0.68 of Thr/Lys in high Lys diet). Body weight gain (BWG) increased in chickens receiving high Lys diet as compared with those fed with the control diet (P<0.05); no significant difference was observed in BWG of chickens from high Lys + Thr and high Lys groups. Feed conversion ratio (FCR) was lower in chickens fed with high Lys or high Lys + Thr diet than in those on the control diet. Serotonin concentration increased in the plasma of chickens fed with high Lys diet as compared to those fed with other diets. A negative correlation was observed between plasma serotonin concentration and FCR. These results provide the first evidence on the use of high Lys in broiler diets to reduce FCR during finisher period, which may be associated with change in plasma serotonin concentration. These findings suggest that high Lys content in finisher diet, but not high Thr + Lys diet, may affect the peripheral serotonergic metabolism and improve FCR. Thus, plasma serotonin may serve as a biomarker of FCR in broilers.

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin⁻chaperonin system.

The phosphole ring is known as a useful building block for constructing π-conjugated organic materials. Here, we report ladder-type benzophospholo[3,2-b]indole (BPI) derivatives where the phosphole and the pyrrole rings are directly fused. Compounds 8a-8d with different aryl groups on the phosphorous center were successfully synthesized, and the solid-state structure of 8a was confirmed using X-ray crystallographic analysis. The BPIs exhibit relatively high fluorescence quantum yield (Φ 0.50-0.72) and demonstrate a larger Stokes shift compared with a series of benzophospholo[3,2-b]benzoheteroles. The benzophospholo[3,2-b]carbazole derivative 9, which possesses a benzene ring between the phosphole and the pyrrole rings of the BPI, was also synthesized, and its solid-state structure was confirmed using X-ray crystallographic analysis. Compound 9 was found to show a smaller Stokes shift compared with the BPI.

Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.

The kelulut bee (Meliponini) is a subfamily of stingless bees that produce honey. A total of 89 species out of a total of 500 species of kelulut bees are known to originate from the Indo-Australian region. Kelulut bees do not have quality standards so they still refer to the Codex and EU Directive which basically only applied for Apis honey. The Codex and EU Directive are formed by several psychochemical parameters, one of it is diastase activity. Diastase activity in kelulut honey is known not to meet existing standards or even undetectable. Therefore, this study aimed to explore proteins inside kelulut honey and investigate the possibility of using a specific protein as a biomarker to differentiate honey produced by kelulut bee from other honey. This research can also be considered as an initial step to optimize the exploration of protein in kelulut honey. This research is divided into two sections which are the preliminary research and the research expansion. From preliminary section, glucose dehydrogenase enzyme (GDH) was found to be present inside Tetragonula spp honey. A further examination of GDH enzyme was made in four kelulut bee honeys namely Tetragonula leaviceps, T. biroi, Heterotrigona itama, and Geniotrigona thoracica. The preliminary research has five stages that are exactly the as expansion research section except it didn't include GDH activity measurement. The research includes seven main stages. First honeys were dialyzed to remove the sugar content followed by centrifugation. The samples were then purified using liquid chromatography with anion exchanger column. The molecular weight of proteins was analysed by SDS-PAGE method. The GDH activity was measured using spectrophotometer followed by qualitative analysis using LC-MS/MS. The peptide sequences resulted from LC-MS/MS were then matched with Uniprot to identify the unknow protein. The results showed that only T. biroi and T. laeviceps had GDH enzyme activity of 0,1891 U/mL and 0,1652-1,579 U/mL, respectively. Bands from both species were also qualitatively identified as GDH. With these results, it can be concluded that the GDH enzyme cannot be used as a biomarker to distinguish the kelulut honey.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.