Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.     

P-31 in-vivo NMR investigation on the function of polyphosphates as phosphate-and energysource during the regreening of the green alga Chlorella fusca

The green alga Chlorella fusca accumulates polyphosphates under conditions of nitrogen starvation while deassembling the photosynthetic apparatus. The polyphosphate content of cells regreening after resupply with nitrate under different culture conditions was investigated by P-31 in-vivo NMR spectroscopy. Neither phosphate deficiency nor anaerobiosis during the first hours of regreening inhibited the recovery of the cells. Polyphosphates were degraded during regeening. Differences in the amount of polyphosphates of phosphate supplied and deficient cells occurred only after more then 8 h. After 16 h phosphate deficient cells had still 75% of the polyphosphate content of phosphate suppled cells. In cells kept under anaerobic conditions polyphosphate degradation was much higher than in oxygen supplied cells. After 8 h they contained less than 50% of the polyphosphate content of oxygen supplied cells. These data suggest that polyphosphates serve as obligatory phosphate source during regreening and may be used as an energy source.Non standard abbreviations EDTA Ethylene diamine tetraacetic acid - FID Free induction decay - MOPSO 3-(N-morpholine)-2-hydroxy-propanesulfonic acid - NMR Nuclear magnetic resonance - PP Polyphosphates - PP₄ central phosphate groups of polyphosphates     

The origin and functional transition of P34

P34, a storage protein and major soybean allergen, has undergone a functional transition from a cysteine peptidase to a syringolide receptor. An exploration of the evolutionary mechanism of this functional transition is made. To identify homologous genes of P34, syntenic network was constructed using syntenic relationships from the Plant Genome Duplication Database. The collected homologous genes, along with SPE31, a highly homologous protein to P34 from the seeds of Pachyrhizus erosus, were used to construct a phylogenetic tree. The results show that multiple gene duplications, exon shuffling and following granulin domain loss and some critical point mutations are associated with the functional transition. Although some tests suggested the existence of positive selection, the possibility that random fixation under relaxation of purifying selection results in the functional transition is also supported. In addition, the genes Glyma08g12340 and Medtr8g086470 may belong to a new group within the papain family.     

Irradiation to the young mouse brain impaired white matter growth more in females than in males

Modern therapy cures 80% of all children with brains tumors, but may also cause long-lasting side effects, so called late effects. Radiotherapy is particularly prone to cause severe late effects, such as intellectual impairment. The extent and nature of the resulting cognitive deficits may be influenced by age, treatment and gender, where girls suffer more severe late effects than boys. The reason for this difference between boys and girls is unknown, but very few experimental studies have addressed this issue. Our aim was to investigate the effects of ionizing radiation on the corpus callosum (CC) in both male and female mice. We found that a single dose of 8 Gray (Gy) to the brains of postnatal day 14 mice induced apoptosis in the CC and reduced the number of proliferating cells by one third, as judged by the number of phospho-histone H3 positive cells 6 h after irradiation (IR). BrdU incorporation was reduced (62% and 42% lower in females and males, respectively) and the number of oligodendrocytes (Olig2⁺ cells) was lower (43% and 21% fewer in females and males, respectively) 4 months after IR, so the lack of developing and differentiated cells was more pronounced in females. The number of microglia was unchanged in females but increased in males at this late time point. The density of microvessel profiles was unchanged by IR. This single, moderate dose of 8 Gy impaired the brain growth to some extent (8.1% and 0.4% lower brain/body weight ratio in females and males, respectively) but the CC growth was even more impaired (31% and 19% smaller in females and males, respectively) 4 months after IR compared with non-irradiated mice. In conclusion, this is the first study to our knowledge demonstrating that IR to the young rodent brain affects white matter development more in females than in males.     

13C-31P Couplings in the Study of Conformational Properties of Some Diastereomeric Nucleoside-3′-Thiophosphate Derivatives

Synthesis and stereochemical characterization of enantiomerically pure nucleoside-3′-phosphorothioate esters and salts are reported. Vicinal carbon–phosphorus couplings reflect different predominance of the ? conformation in the isomeric (Rp and Sp) esters, while for the salts the ?^t conformation prevails in both stereoisomers. The influence of solvent and temperature on the conformational preferences is also described.     

Vectorial redox reactions of physiological quinones. II. A study of transient semiquinone formation

Transient absorption changes during reduction of quinone in liposomes by external dithionite, in the absence and presence of initially trapped ferricyanide, were matched with absorption spectra of semiquinone and quinone in the blue region. Plastoquinone, ubiquinone-9 and phylloquinone, each having an isoprenoid side chain were compared with trimethyl-p-benzoquinone, ubiquinone-9 and menadione, which lack a long side chain.Semiquinone transients could only be observed by our spectroscopic technique during reduction of quinones lacking the chain. If Triton X-100 was added to the liposomes preparation semiquinone transients were also observed with the isoprenoid quinones. This result is consistent with the view that isoprenoid quinones build domains in the membranes, in which the life time of the semiquinone might be decreased by fast disproportionation, and to which dithionite has limited access.