Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases

Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD(+), suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.     

Coordinated reversal of flagellar motors on a single Escherichia coli cell

An Escherichia coli cell transduces extracellular stimuli sensed by chemoreceptors to the state of an intracellular signal molecule, which regulates the switching of the rotational direction of the flagellar motors from counterclockwise (CCW) to clockwise (CW) and from CW back to CCW. Here, we performed high-speed imaging of flagellar motor rotation and show that the switching of two different motors on a cell is controlled coordinatedly by an intracellular signal protein, phosphorylated CheY (CheY-P). The switching is highly coordinated with a subsecond delay between motors in clear correlation with the distance of each motor from the chemoreceptor patch localized at a cell pole, which would be explained by the diffusive motion of CheY-P molecules in the cell. The coordinated switching becomes disordered by the expression of a constitutively active CheY mutant that mimics the CW-rotation stimulating function. The coordinated switching requires CheZ, which is the phosphatase for CheY-P. Our results suggest that a transient increase and decrease in the concentration of CheY-P caused by a spontaneous burst of its production by the chemoreceptor patch followed by its dephosphorylation by CheZ, which is probably a wavelike propagation in a subsecond timescale, triggers and regulates the coordinated switching of flagellar motors.     

Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes

Skin color is one of the most important fruit traits in grape, and has become greatly diversified due to hybridization and human selection. Many studies concerning the genetic control of grape color in European species (Vitis vinifera L.), especially the role of MYB-related genes, have been reported. On the other hand, there have been few studies of the MYB-related genes in grapes belonging to V.?×labruscana L.H. Bailey, a subgroup of grapes that originated from the hybridization of V. labrusca with V. vinifera. In the present study, we found a novel functional haplotype, HapE2 (consisting of the genes VlMYBA2 and VlMYBA1?C3), in diploid V.?×labruscana. Moreover, we developed a method to determine the haplotype compositions of tetraploid grapes by means of quantitative real-time PCR, and investigated the relationship between haplotype composition and skin color. The color locus in V.?×labruscana grapes usually consists of functional haplotypes (HapE1 and/or HapE2), and non-functional haplotype HapA. The number of functional haplotypes in the genome was found to be correlated with the level of anthocyanin in the skin. Anthocyanin contents of grapes that contained HapE2 were significantly higher than those containing HapE1. These results suggest that the number and kind of functional haplotypes at the color locus are the major genetic factors that determine skin color variation. These findings provide new knowledge about the unique genetic control of color in V.?×labruscana grapes, and should contribute to development of new cultivars that have the desired color and anthocyanin content.     

Creation of a cellooligosaccharide-assimilating Escherichia coli strain by displaying active beta-glucosidase on the cell surface via a novel anchor protein

We demonstrated direct assimilation of cellooligosaccharide using Escherichia coli displaying beta-glucosidase (BGL). BGL from Thermobifida fusca YX (Tfu0937) was displayed on the E. coli cell surface using a novel anchor protein named Blc. This strain was grown successfully on 0.2% cellobiose, and the optical density at 600 nm (OD(600)) was 1.05 after 20 h.     

Direct and efficient ethanol production from high-yielding rice using a Saccharomyces cerevisiae strain that express amylases

Efficient ethanol producing yeast Saccharomyces cerevisiae cannot produce ethanol from raw starch directly. Thus the conventional ethanol production required expensive and complex process. In this study, we developed a direct and efficient ethanol production process from high-yielding rice harvested in Japan by using amylase expressing yeast without any pretreatment or addition of enzymes or nutrients. Ethanol productivity from high-yielding brown rice (1.1g/L/h) was about 5-fold higher than that obtained from purified raw corn starch (0.2g/L/h) when nutrients were added. Using an inoculum volume equivalent to 10% of the fermentation volume without any nutrient supplementation resulted in ethanol productivity and yield reaching 1.2g/L/h and 101%, respectively, in a 24-h period. High-yielding rice was demonstrated to be a suitable feedstock for bioethanol production. In addition, our polyploid amylase-expressing yeast was sufficiently robust to produce ethanol efficiently from real biomass. This is first report of direct ethanol production on real biomass using an amylase-expressing yeast strain without any pretreatment or commercial enzyme addition.     

Protective roles of CX3CR1-mediated signals in toxin A-induced enteritis through the induction of heme oxygenase-1 expression

The injection of Clostridium difficile toxin A into the ileal loops caused fluid accumulation with the destruction of intestinal epithelial structure and the recruitment of neutrophils and macrophages. Concomitantly, intraileal gene expression of CX3CL1/fractalkine (FKN) and its receptor, CX3CR1, was enhanced. When treated with toxin A in a similar manner, CX3CR1-deficient (CX3CR1(-/-)) mice exhibited exaggerated fluid accumulation, histopathological alterations, and neutrophil recruitment, but not macrophage infiltration. Mice reconstituted with CX3CR1(-/-) mouse-derived bone marrow cells exhibited exacerbated toxin A-induced enteritis, indicating that the lack of the CX3CR1 gene for hematopoietic cells aggravated toxin A-induced enteritis. A heme oxygenase-1 (HO-1) inhibitor, tin-protoporphyrin-IX, markedly increased fluid accumulation in toxin A-treated wild-type mice, indicating the protective roles of HO-1 in this situation. HO-1 expression was detected mainly in F4/80-positive cells expressing CX3CR1, and CX3CR1(-/-) mice failed to increase HO-1 expression after toxin A treatment. Moreover, CX3CL1/FKN induced HO-1 gene expression by isolated lamina propria-derived macrophages or a mouse macrophage cell line, RAW264.7, through the activation of the ERK signal pathway. Thus, CX3CL1/FKN could induce CX3CR1-expressing macrophages to express HO-1, thereby ameliorating toxin A-induced enteritis.     

Urinary 8-OHdG elevations in a partial lesion rat model of Parkinson's disease correlate with behavioral symptoms and nigrostriatal dopaminergic depletion

Increased oxidative stress contributes to pathogenesis of Parkinson's disease (PD). 8-hydroxy-2'-deoxyguanosine (8-OHdG) is the oxidation product most frequently measured as an indicator of oxidative DNA damage. Several studies have shown increased 8-OHdG in PD patients. There are few basic laboratory data examining 8-OHdG levels in animal models of PD. In this study, we utilized hemiparkinsonian model of rats induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). The urinary 8-OHdG level was measured in relation to behavioral and pathological deficits arising from 6-OHDA-induced neurotoxic effects on the nigrostriatal dopaminergic pathway. All rats were subjected to a series of behavioral tests for 42 days after 6-OHDA injection. We collected urine samples with subsequent measurement of 8-OHdG level using ELISA kits. For immunohistochemical evaluation, tyrosine hydroxylase (TH) staining was performed. Significant increments in urinary 8-OHdG level were observed continuously from day 7 until day 35 compared to control group, which showed a trend of elevation as early as day 3. Such elevated urinary 8-OHdG level significantly correlated with all of the behavioral deficits measured here, suggesting that urinary 8-OHdG level provides a good index of severity of parkinsonism. Urinary 8-OHdG level also had a significant positive correlation with the survival rate of dopaminergic fibers or neurons, advancing the concept that oxidative stress during the early phase of 6-OHDA neurotoxicity may correspond to disease progression closely approximating neuronal degeneration in the nigrostriatal dopaminergic system. The present results demonstrate that alterations in urinary 8-OHdG level closely approximate onset and disease progression in PD.     

Enzymatic glutathione production using metabolically engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> as a whole-cell biocatalyst

We developed a novel enzymatic glutathione (GSH) production system using Saccharomyces cerevisiae as a whole-cell biocatalyst, and improved its GSH productivity by metabolic engineering. We demonstrated that the metabolic engineering of GSH pathway and ATP regeneration can significantly improve GSH productivity by up to 1.7-fold higher compared with the parental strain, respectively. Furthermore, the combination of both improvements in GSH pathway and ATP regeneration is more effective (2.6-fold) than either improvement individually for GSH enzymatic production using yeast. The improved whole-cell biocatalyst indicates its great potential for applications to other kinds of ATP-dependent bioproduction.     

Neuronal suppressor of cytokine signaling-3 deficiency enhances hypothalamic leptin-dependent phosphatidylinositol 3-kinase signaling

Suppressor of cytokine signaling-3 (SOCS3) is thought to be involved in the development of central leptin resistance and obesity by inhibiting STAT3 pathway. Because phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in transducing leptin action in the hypothalamus, we examined whether SOCS3 exerted an inhibition on this pathway. We first determined whether leptin sensitivity in the hypothalamic PI3K pathway was increased in brain-specific Socs3-deficient (NesKO) mice. In NesKO mice, hypothalamic insulin receptor substrate-1 (IRS1)-associated PI3K activity was significantly increased at 30 min and remained elevated up to 2 h after leptin intraperitoneal injection, but in wild-type (WT) littermates, the significant increase was only at 30 min. Hypothalamic p-STAT3 levels were increased up to 5 h in NesKO as opposed to 2 h in WT mice. In food-restricted WT mice with reduced body weight, leptin increased hypothalamic PI3K activity only at 30 min, and p-STAT3 levels at 30-120 min postinjection. These results suggest increased leptin sensitivity in both PI3K and STAT3 pathways in the hypothalamus of NesKO mice, which was not due to a lean phenotype. In the next experiment with a clonal hypothalamic neuronal cell line expressing proopiomelanocortin, we observed that whereas leptin significantly increased IRS1-associated PI3K activity and p-JAK2 levels in cells transfected with control vector, it failed to do so in SOCS3-overexpressed cells. Altogether, these results imply a SOCS3 inhibition of the PI3K pathway of leptin signaling in the hypothalamus, which may be one of the mechanisms behind the development of central leptin resistance and obesity.