Sulerythrin,the smallest member of the rubrerythrin family,from a strictly aerobic and thermoacidophilic archaeon,Sulfolobus tokodaii strain 7

A protein corresponding to the N-terminal domain of rubrerythrin was isolated from a strictly aerobic archaeon, Sulfolobus tokodaii strain 7. The molecular mass was found to be 15.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 16278 Da by time-of-flight mass spectrometry and 34.5 kDa by gel filtration chromatography, suggesting that the protein is dimeric. Two mol iron and 1-2 mol zinc mol(-1) protein were detected. On addition of the azide ion, the absorption spectrum was greatly affected. The far UV circular dichroism spectrum suggested that the protein was mostly composed of alpha-helices. The N-terminal sequence completely matched the open reading frame, st2370, recently found on genome analysis of the organism. The protein was homologous to rubrerythrin but lacked a C-terminal rubredoxin domain. It was found in the genus Sulfolobus and therefore named sulerythrin; it is the smallest and first aerobic member of the rubrerythrin family.     

Crystal structure of sulerythrin, a rubrerythrin-like protein from a strictly aerobic archaeon, Sulfolobus tokodaii strain 7, shows unexpected domain swapping

Sulerythrin is the first rubrerythrin-like protein to be isolated from an aerobic organism, Sulfolobus tokodaii strain 7, and it lacks a C-terminal rubredoxin-like FeS(4) domain. The protein purified from Sulfolobus cells was crystallized, and the crystal structure was determined at 1.7 A resolution. The dimer of sulerythrin exhibited "domain-swapping" at the loop connecting alphaB and alphaC, hybrid four-helix bundles consisting of alphaA/B and alphaC/D being formed. The structure and atomic identity of the binuclear metal center were determined by means of anomalous scattering analysis. The site contained 1.0 mol of hexacoordinate Fe, 0.80-0.87 mol of tetracoordinate Zn, and 0.73-0.88 mol of putative O(2) per monomer. The metal ions were found at exchanged positions compared to those in the Fe/Zn-containing rubrerythrin from Desulfovibrio vulgaris. The results demonstrate that the binuclear metal center of rubrerythrin-like proteins is plastic in its ability to bind metal ions.     

Effect of solvent exchange on the solid structure and dissolution behavior of cellulose

Effects of solvent exchange and milling on the solid structure of cellulose were investigated, using small- and wide-angle X-ray scattering and solid-state NMR. The solvent exchange facilitated the dissolution of cellulose in LiCl/DMAc with no change of the crystalline structure of cellulose. In contrast, the milling never facilitated the dissolution of cellulose, though the crystalline structure was almost destroyed. These facts show that the crystalline structure of cellulose hardly affects the dissolution in LiCl/DMAc. The fractal dimensions determined by the small-angle X-ray scattering measurements were increased by the solvent exchange, suggesting that the aggregation state of the cellulose microfibril is affected. It was also suggested by the NMR (1)H spin relaxation time measurements that the solvent exchange enhances the molecular mobility of cellulose and shortens the characteristic length along the microfibril, which allows easier access of the solvent molecule to cellulose.     

Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Structural basis of compound recognition by adenosine deaminase

Structural snapshots corresponding to various states enable elucidation of the molecular recognition mechanism of enzymes. Adenosine deaminase has two distinct conformations, an open form and a closed form, although it has so far been unclear what factors influence adaptation of the alternative conformations. Herein, we have determined the first nonligated structure as an initial state, which was the open form, and have thereby rationally deduced the molecular recognition mechanism. Inspection of the active site in the nonligated and ligated states indicated that occupancy at one of the water-binding positions in the nonligated state was highly significant in determining alternate conformations. When this position is empty, subsequent movement of Phe65 toward the space induces the closed form. On the other hand, while occupied, the overall conformation remains in the open form. This structural understanding should greatly assist structure-oriented drug design and enable control of the enzymatic activity.     

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues

We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named "HS-ovalbumin" in the present study, was found to have a T(m) value of 83 degrees C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.     

Genotoxicity of microcystin-LR in human lymphoblastoid TK6 cells

Toxic cyanobacteria (blue-green algae) water blooms have become a serious problem in several industrialized areas of the world. Microcystin-LR (MCLR) is a cyclic heptapeptidic toxin produced by the cyanobacteria. In the present study, we used human lymphoblastoid cell line TK6 to investigate the in vitro genotoxicity of MCLR. In a standard 4h treatment, MCLR did not induce a significant cytotoxic response at <80 microg/ml. In a prolonged 24h treatment, in contrast, it induced cytotoxic as well as mutagenic responses concentration-dependently starting at 20 microg/ml. At the maximum concentration (80 microg/ml), the micronucleus frequency and the mutation frequency at the heterozygous thymidine kinase (TK) locus were approximately five-times the control values. Molecular analysis of the TK mutants revealed that MCLR specifically induced loss of heterozygosity at the TK locus, but not point mutations or other small structural changes. These results indicate that MCLR had a clastogenic effect. We discuss the mechanisms of MCLR genotoxicity and the possibility of its being a hepatocarcinogen.