Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23     

Biosynthesis of linoleic acid in Tyrophagus mites (Acarina: Acaridae)

We report here that Tyrophagus similis and Tyrophagus putrescentiae (Astigmata: Acaridae) have the ability to biosynthesize linoleic acid [(9Z, 12Z)-9, 12-octadecadienoic acid] via a Δ12-desaturation step, although animals in general and vertebrates in particular appear to lack this ability. When the mites were fed on dried yeast enriched with d₃₁-hexadecanoic acid (16:0), d₂₇-octadecadienoic acid (18:2), produced from d₃₁-hexadecanoic acid through elongation and desaturation reactions, was identified as a major fatty acid component of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in the mites. The double bond position of d₂₇-octadecadienoic acid (18:2) of PCs and PEs was determined to be 9 and 12, respectively by dimethyldisulfide (DMDS) derivatization. Furthermore, the GC/MS retention time of methyl 9, 12-octadecadienoate obtained from mite extracts agreed well with those of authentic linoleic acid methyl ester. It is still unclear whether the mites themselves or symbiotic microorganisms are responsible for inserting a double bond into the Δ12 position of octadecanoic acid. However, we present here the unique metabolism of fatty acids in the mites.     

Recombinant production and characterization of an N-acyl-d-amino acid amidohydrolase from Streptomyces sp. 64E6

N-Acyl-d-amino acid amidohydrolases (d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)d-Phe (0.05–6.32 μmol min^?1 mg^?1) under conditions without CoCl₂. Addition of 1 mM CoCl₂ enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min^?1 mg^?1 for N-Ac-d-Phe and N-Ac-d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0–9.0. It was stable at pH 5.5–9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-d-Phe was 2.1-fold higher than that toward N-Ac-d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.     

Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M–1

Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 nm. The enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the protein. The enzyme was inactivated by Ag⁺, Hg²⁺, Zn²⁺ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines. Km and Vmax values for dimethylglycine were 9.1 mm and 1.22 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Dimethylglycine+O₂+H₂O → sarcosine+formaldehyde+H₂O₂.     

Lung Surfactant Levels are Regulated by Ig-Hepta/GPR116 by Monitoring Surfactant Protein D

Lung surfactant is a complex mixture of lipids and proteins, which is secreted from the alveolar type II epithelial cell and coats the surface of alveoli as a thin layer. It plays a crucial role in the prevention of alveolar collapse through its ability to reduce surface tension. Under normal conditions, surfactant homeostasis is maintained by balancing its release and the uptake by the type II cell for recycling and the internalization by alveolar macrophages for degradation. Little is known about how the surfactant pool is monitored and regulated. Here we show, by an analysis of gene-targeted mice exhibiting massive accumulation of surfactant, that Ig-Hepta/GPR116, an orphan receptor, is expressed on the type II cell and sensing the amount of surfactant by monitoring one of its protein components, surfactant protein D, and its deletion results in a pulmonary alveolar proteinosis and emphysema-like pathology. By a coexpression experiment with Sp-D and the extracellular region of Ig-Hepta/GPR116 followed by immunoprecipitation, we identified Sp-D as the ligand of Ig-Hepta/GPR116. Analyses of surfactant metabolism in Ig-Hepta^+/+ and Ig-Hepta^−/− mice by using radioactive tracers indicated that the Ig-Hepta/GPR116 signaling system exerts attenuating effects on (i) balanced synthesis of surfactant lipids and proteins and (ii) surfactant secretion, and (iii) a stimulating effect on recycling (uptake) in response to elevated levels of Sp-D in alveolar space.     

Large-scale profiling of brown rice ionome in an ethyl methanesulphonate-mutagenized hitomebore population and identification of high- and low-cadmium lines

Background and aim

Recycled sources of phosphorus (P), such as struvite extracted from wastewater, have potential to substitute for more soluble manufactured fertilisers and help reduce the long-term threat to food security from dwindling finite reserves of phosphate rock (PR). This study aimed to determine whether struvite could be a component of a sustainable P fertiliser management strategy for arable crops.

Methods

A combination of laboratory experiments, pot trials and mathematical modelling of the root system examined the P release properties of commercial fertiliser-grade struvite and patterns of P uptake from a low-P sandy soil by two different crop types, in comparison to more soluble inorganic P fertilisers (di-ammonium phosphate (DAP) and triple super phosphate (TSP)).

Results

Struvite had greatly enhanced solubility in the presence of organic acid anions; buckwheat, which exudes a high level of organic acids, was more effective at mobilising struvite P than the low level exuder, spring wheat. Struvite granules placed with the seed did not provide the same rate of P supply as placed DAP granules for early growth of spring wheat, but gave equivalent rates of P uptake, yield and apparent fertiliser recovery at harvest, even though only 26 % of struvite granules completely dissolved. Fertiliser mixes containing struvite and DAP applied to spring wheat have potential to provide both optimal early and late season P uptake and improve overall P use efficiency.

Conclusions

We conclude that the potential resource savings and potential efficiency benefits of utilising a recycled slow release fertiliser like struvite offers a more sustainable alternative to only using conventional, high solubility, PR-based fertilisers.

     

Isolation and characterization of a novel mycovirus infecting an edible mushroom,Grifola frondosa

Grifola frondosa (Maitake mushroom) is an important cultivated mushroom due to its medicinal and nutrient values. In this study, we isolated and characterized a novel partitivirus (named Grifola frondosa partitivirus 1, GfPV1) infecting a standard G. frondosa strain Gf-N2. This virus has a two-segmented dsRNA genome (dsRNA1 and dsRNA2) with nucleotide lengths of 2.3 and 2.2 kbp, respectively. The coding strand of dsRNA1 and dsRNA2 segments carries single open reading frame encoding RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. BLAST searches and phylogenetic analyses showed that GfPV1 is most closely related to a betapartitivirus, Lentinula edodes partitivirus 1 (RdRp <70% and CP <60% amino acid sequence identities), but the sequence divergence suggests that GfPV1 is classifiable as a new member of the genus Betapartitivirus, family Partitiviridae. The presence of GfPV1 does not affect colony morphology and fruiting body development of G. frondosa. This is the first report investigating the effects of a mycovirus infection on the colony morphology and fruiting body development of G. frondosa. Interestingly, GfPV1 accumulations markedly decreased along with the fruiting body maturation stages, suggesting the inhibition of virus multiplication during sexual phase of the G. frondosa life cycle.