Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Caecal environment of rats fed far East Asian-modelled diets

To clarify the effect of type of foods on the intestinal environment, Far East Asian- (FEA; rich in rice starch, soy protein and soy oil) and Far East Asian marine- (FEAM; rich in rice starch, fish meal, fish oil and brown alga) modelled diets and sucrose, casein and beef tallow-rich (SCB) diet were prepared. After the 2-week administration of diets in rats, caecal organic acids and putrefactive compounds (ammonia, indole, phenol and H₂S, which are regarded as putative risk factors for tumours) were determined. The caecal microbiota was also analyzed using denaturing gradient gel electrophoresis and pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. Levels of n-butyrate, acetate, indole and phenol were high in rats fed FEA. On the other hand, H₂S was clearly suppressed by both FEA and FEAM comparing with SCB. These results suggest that FEAM is preferable to FEA for optimal intestinal environment and host health. Both microbial analyses showed that the diversity of microbiota in the FEAM group was lower than in the other diet groups. Ratio of Firmicutes, Bacteroidetes and Proteobacteria in the SCB group was about 5:4:1. Firmicutes, particularly Lachnospiraceae, was promoted by FEA and FEAM.     

SAPl8和Sufu蛋白质复合物的表达纯化研究

通过分子克隆技术将Sufu和SAP18构建到原核载体,在原核表达系统进行外源表达,采用亲和层析和分子筛层析对Sufu和SAPl8及其复合物进行纯化。同时利用非变性胶的方法进一步确定该蛋白之间的相互作用及结合比例。结果表明,原核表达系统中Sufu和SAPl8蛋白表达量高,可以纯化到高纯度的蛋白质。su如和SAPl8结合的摩尔比为1：1,按摩尔比1：2混合、纯化可以得到高纯度、稳定的蛋白复合物,从而为进一步复合物的结构生物学研究奠定基础。     

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy–Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.     

The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum

Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na⁺/H⁺ antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum ‘Jinba’. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na⁺ into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K⁺/Na⁺ ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na⁺ accumulation. This study is the first to report the transgenic expression of a Na⁺ efflux carrier in chrysanthemum.     

Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein

P_II protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many P_II proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, P_II protein from Synechococcus elongatus, a model for cyanobacteria and plant P_II proteins, is utilized to reveal the anticooperative mechanism upon binding of 2‐oxoglutarate (2‐OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2‐OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T‐loop, which is believed to be responsible for mediating the binding of P_II with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of P_II variants further confirmed the influence of T‐loop on the anticooperative regulation, especially on binding of the third 2‐OG. These discoveries extend our understanding of the P_II T‐loop from being essential in versatile binding of target protein to signal‐mediating in the anticooperative allosteric regulation. Proteins 2014; 82:1048–1059. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk

Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.     

Synthesis of Novel Chiral Tridentate Schiff‐Base Ligands and Their Applications in Catalytic Asymmetric Henry Reaction

A series of chiral tridentate Schiff‐bases were prepared and used as ligands in the catalytic asymmetric Henry reaction. Under the optimal conditions, a variety of arylaldehydes were smoothly converted into corresponding adducts with high yields (up to 98%) and excellent enantioselectivities (up to 97% ee). Chirality 26: 780–783, 2014. © 2014 Wiley Periodicals, Inc.     

Crystal structure of an HD‐GYP domain cyclic‐di‐GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

Bis‐(3′,5′) cyclic di‐guanylate (c‐di‐GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c‐di‐GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD‐GYP domains. Here, we have determined the structure of an enzymatically active HD‐GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c‐di‐GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c‐di‐GMP. This structure completes the picture of all domains involved in c‐di‐GMP metabolism and reveals that the HD‐GYP family splits into two distinct subgroups containing bi‐ and trinuclear metal centres.