Phosphorylation of phenol by phenylphosphate synthase: role of histidine phosphate in catalysis

The anaerobic metabolism of phenol proceeds via carboxylation to 4-hydroxybenzoate by a two-step process involving seven proteins and two enzymes ("biological Kolbe-Schmitt carboxylation"). MgATP-dependent phosphorylation of phenol catalyzed by phenylphosphate synthase is followed by phenylphosphate carboxylation. Phenylphosphate synthase shows similarities to phosphoenolpyruvate (PEP) synthase and was studied for the bacterium Thauera aromatica. It consists of three proteins and transfers the beta-phosphoryl from ATP to phenol; the products are phenylphosphate, AMP, and phosphate. We showed that protein 1 becomes phosphorylated in the course of the reaction cycle by [beta-(32)P]ATP. This reaction requires protein 2 and is severalfold stimulated by protein 3. Stimulation of the reaction by 1 M sucrose is probably due to stabilization of the protein(s). Phosphorylated protein 1 transfers the phosphoryl group to phenolic substrates. The primary structure of protein 1 was analyzed by nanoelectrospray mass spectrometry after CNBr cleavage, trypsin digestion, and online high-pressure liquid chromatography at alkaline pH. His-569 was identified as the phosphorylated amino acid. We propose a catalytic ping-pong mechanism similar to that of PEP synthase. First, a diphosphoryl group is transferred to His-569 in protein 1, from which phosphate is cleaved to render the reaction unidirectional. Histidine phosphate subsequently serves as the actual phosphorylation agent.     

Immunolocalisation of two tropinone reductases in potato (Solanum tuberosum L.) root, stolon, and tuber sprouts

Tropinone reductases (TRs) are essential enzymes in the tropane alkaloid biosynthesis, providing either tropine for hyoscyamine and scopolamine formation or providing pseudotropine for calystegines. Two cDNAs coding for TRs were isolated from potato (Solanum tuberosum L.) tuber sprouts and expressed in E. coli. One reductase formed pseudotropine, the other formed tropine and showed kinetic properties typical for tropine-forming tropinone reductases (TRI) involved in hyoscyamine formation. Hyoscyamine and tropine are not found in S. tuberosum plants. Potatoes contain calystegines as the only products of the tropane alkaloid pathway. Polyclonal antibodies raised against both enzymes were purified to exclude cross reactions and were used for Western-blot analysis and immunolocalisation. The TRI (EC 1.1.1.206) was detected in protein extracts of tuber tissues, but mostly in levels too low to be localised in individual cells. The function of this enzyme in potato that does not form hyoscyamine is not clear. The pseudotropine-forming tropinone reductase (EC 1.1.1.236) was detected in potato roots, stolons, and tuber sprouts. Cortex cells of root and stolon contained the protein; additional strong immuno-labelling was located in phloem parenchyma. In tuber spouts, however, the protein was detected in companion cells.     

Effect of short-term starvation of adult Antarctic krill, Euphausia superba, at the onset of summer

We investigated the effect of short-term starvation (18 days) on the physiology of adult Euphausia superba from the Lazarev Sea at the onset of summer. Metabolic data, elemental and biochemical composition as well as morphological parameters revealed that, at the beginning of the experiment, krill was in transition from winter to summer physiology, with some features typical for late winter/spring (low lipid reserves, low C:N ratio, elevated O:N ratio) and others for summer (high respiration rates, high MDH activity, large green digestive gland, short intermoult period (IMP) and fast growth).Starvation reduced body reserves drastically by more than 1% C per day. In relative terms, lipids (40%) and glycogen (30%) were reduced most and proteins by 10% of the initial value. Absolute consumption, however, was approximately 4% DM for lipids and proteins each, whereas contribution of glycogen was negligible. Within lipids, triacylglycerols (TAG) and phospholipids (PL) fell most dramatically from already low levels by 84% and 39%, respectively. Phosphatylcholine (PC) constituted 57% of PL and declined by 46%. As a result, proportions of the lipid classes changed with sterols increasing relatively. Metabolite changes were similar in cephalothorax and abdomen, although TAG in the cephalothorax fell more drastically. High metabolic activity at the beginning of starvation was quickly reduced to reach 53% after 18 days, accompanied by a reduction in the abdominal activity of malate dehydrogenase (MDH) by 25%. Our results may provide some explanation why recruitment of some year-classes of krill fails.Despite execution of the experiment in spring (i.e. transitional physiology state) and its short duration, some changes in the activity of metabolic enzymes in the abdomen, representing lipolytic, glycolytic and proteolytic pathways, respectively, were measured. Rising activities of 3-hydroxyacyl-CoA dehydrogenase (HOAD) and glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) indicated increased lipolytic and glycolytic fluxes, respectively, whereas declining glutamate dehydrogenase (GluDH) activity suggests reduced proteolytic flux. Activities of other enzymes from protein catabolism, alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT), however, remained unchanged. Ratios calculated from these trends indicated a declining importance of protein use during the course of starvation compared with consumption of lipids and glycogen. These results suggest that constant-proportion enzymes from different catabolic pathways, and calculated ratios thereof, may be useful in detecting shifts between the consumption of different body reserves.     

Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QB in photosystem II

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q_B to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG_L) enhances the affinity for BQ that inactivates Q_B. 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q_B during 10 days of PG depletion, and was recovered rapidly within 10 h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG_H) displaces Q_B directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14 days decreased the chlorophyll/PBS ratio to about 1/4, florescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683 nm with a long 1.2 ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG_H, PG_L and other PG molecules on PSII structure are discussed.     

The activity ratio of 228Th to 228Ra in bone tissue of recently deceased humans: a new dating method in forensic examinations

Reliable determination of time since death in human skeletons or single bones often is limited by methodically difficulties. Determination of the specific activity ratio of natural radionuclides, in particular of 232Th (Thorium), 228Th and 228Ra (Radium) seems to be a new appropriate method to calculate the post mortem interval. These radionuclides are incorporated by any human being, mainly from food. So with an individual's death the uptake of radionuclides ends. But the decay of 232Th produces 228Ra and 228Th due to its decay series, whereas 228Th is continuously built up in the human's bones. Thus, it can be concluded that in all deceased humans at different times after death different activity ratios of 228Th to 228Ra will develop in bone. According to this fact it should be possible to calculate time since death of an individual by first analysing the specific activities of 228Th and 228Ra in bones of deceased and then determining the 228Th/228Ra activity ratio, which can be assigned to a certain post-mortem interval.     

Reduced male fertility is common but highly variable in form and severity in a natural house mouse hybrid zone

Barriers to gene flow between naturally hybridizing taxa reveal the initial stages of speciation. Reduced hybrid fertility is a common feature of reproductive barriers separating recently diverged species. In house mice (Mus musculus), hybrid male sterility has been studied extensively using experimental crosses between subspecies. Here, we present the first detailed picture of hybrid male fertility in the European M. m. domesticus-M. m. musculus hybrid zone. Complete sterility appears rare or absent in natural hybrids but a large proportion of males (~30%) have sperm count or relative testis weight below the range in pure subspecies, and likely suffer reduced fertility. Comparison of a suite of traits related to fertility among subfertile males indicates reduced hybrid fertility in the contact zone is highly variable among individuals and ancestry groups in the type, number, and severity of spermatogenesis defects present. Taken together, these results suggest multiple underlying genetic incompatibilities are segregating in the hybrid zone, which likely contribute to reproductive isolation between subspecies.