Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k_cat/K_m values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.     

Hymenolepis diminuta: Action of worm RNase against RNA

Hymenolepis diminuta possesses a tegumental ribonuclease (RNase) which hydrolyzes rat liver and degraded yeast RNA. Polyacrylamide gel electrophoresis and gel chromatography of rat liver RNA after incubation with intact worms demonstrated significant hydrolysis of the high molecular weight RNA fractions (28 S and 18 S), with the appearance of fractions of intermediate molecular weight (i.e., between 18 S and 4 S), as well as ethanol-soluble fractions. Hydrolysis of degraded yeast RNA (with a molecular weight of approximately 25,000) yielded a single ethanol-precipitable hydrolysis product, as well as ethanol-soluble hydrolysis products.     

Metabolic Reconfiguration in C. elegans Suggests a Pathway for Widespread Sterol Auxotrophy in the Animal Kingdom

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Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.     

Higher order assembling of the mycobacterial polar growth factor DivIVA/Wag31

DivIVA or Wag31, which is an essential pole organizing protein in mycobacteria, can self-assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. The structural organization of polar scaffold formed by polymerization of coiled-coil rich Wag31, which is implicated in the anti-tubercular activities of amino-pyrimidine sulfonamides, remains to be determined. A single-site phosphorylation in Wag31 regulates peptidoglycan biosynthesis in mycobacteria. We report biophysical characterizations of filaments formed by mycobacterial Wag31 using circular dichroism, atomic force microscopy and small angle solution X-ray scattering. Atomic force microscopic images of the wild-type, a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of Wag31 show mostly linear filament formation with occasional curving, kinking and apparent branching. Solution X-ray scattering data indicates that the phospho-mimetic forms of the Wag31 polymers are on average more compact than their phospho-ablative counterparts, which is likely due to the extent of bending/branching. Observed structural features in this first view of Wag31 filaments suggest a basis for higher order Wag31 scaffold formation at the pole.     

Signal transduction in blue light‐induced oxygen uptake in Chlorella cells

In DCMU‐poisoned wild‐type and in non‐photosynthetic pigment mutant cells of Chlorella kessleri , grown heterotrophically with glucose as a carbon source and with nitrate as sole nitrogen source, the known blue light‐enhanced uptake of oxygen and breakdown of starch were reduced by staurosporine and K252a, both potent inhibitors of protein kinase C. This corresponded to sensitivity to these inhibitors of blue light‐induced uptake of nitrate of such organisms. Cells grown with ammonia as sole nitrogen source responded to short wavelength visible irradiation with an increase in oxygen uptake, and this, too, was inhibited by staurosporine and K252a. However, these cells did not show any blue light‐enhanced uptake of nitrate. From these results, enhanced consumption of oxygen under blue light cannot be a consequence of blue light‐induced protein phosphorylation involved in the light‐dependent uptake of nitrate. However, existence of a specific protein phosphorylation within the process of enhancement of oxygen uptake under blue light is not yet proven by the data. There might be a master reaction that induces both processes independently, or there may be influences of other light‐induced processes which lead to enhanced starch breakdown, thereby supplying the glucose for oxidative degradation.     

Identification and in silico prediction of metabolites of tebufenozide derivatives by major human cytochrome P450 isoforms

Cytochrome P450 (CYP) enzymes constitute a superfamily of heme-containing monooxygenases. CYPs are involved in the metabolism of many chemicals such as drugs and agrochemicals. Therefore, examining the metabolic reactions by each CYP isoform is important to elucidate their substrate recognition mechanisms. The clarification of these mechanisms may be useful not only for the development of new drugs and agrochemicals, but also for risk assessment of chemicals. In our previous study, we identified the metabolites of tebufenozide, an insect growth regulator, formed by two human CYP isoforms: CYP3A4 and CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen atom abstraction energy estimation at the density functional theory level, respectively. In this study, the same in silico prediction method was applied to the metabolites of tebufenozide derivatives by major human CYPs (CYP1A2, 2C9, 2C19, 2D6, and 3A4). In addition, the production rate of the metabolites by CYP3A4 was quantitively analyzed by frequency based on docking simulation and hydrogen atom abstraction energy using the classical QSAR approach. Then, the obtained QSAR model was applied to predict the sites of metabolism and the metabolite production order by each CYP isoform.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

Principles of selective inactivation of a viral genome. Comparative kinetic study of modification of the viral RNA and model protein with oligoaziridines

Comparative kinetic analysis of inactivation of bacteriophage MS2 infectivity and aminoalkylation of a model protein (trypsin inhibitor) with oligoaziridines was performed in order to evaluate the selectivity of viral RNA modification with oligocationic reagents. The transition from ethyleneimine monomer to di-, tri-, and tetramer leads to a sharp increase in the rate constant of infectivity inactivation, whereas the rate constant of protein modification changes insignificantly. The selectivity coefficient of the phage RNA aminoalkylation relative to trypsin inhibitor modification increases in this series by more than an order of magnitude. This effect is probably associated with the strengthening of the reagent binding to the nucleic acid, which implies a reaction mechanism that involves the formation of a reactive intermediate. The latter might be an electrostatic complex of the oligocationic reagent and RNA, the only polyanion in the virion. A pronounced decrease in the rate constant of infectivity inactivation in the presence of multiply charged anions (in phosphate buffer) and a biogenic polyamine (spermine) favors this hypothesis. Increasing the reaction temperature increases the rate constant of infectivity inactivation and decreases selectivity of the viral RNA modification.