SummaryGenome scanning of Corynebacterium glutamicum ATCC13032 revealed the presence of five different genes encoding enzymes belonging to three putative trehalose biosynthesis pathways (OtsAB, TreYZ, TreS). The function of the different pathways and of trehalose as an osmoprotectant was studied by characterizing several strains defective for individual trehalose biosynthetic routes. Trehalose synthesis was shown to increase upon hyperosmotic conditions. Cytoplasmic trehalose levels varied considerably depending on kind and accessibility of carbon and nitrogen sources. In contrast to other organisms, osmoregulated trehalose synthesis in C. glutamicum is mediated by the TreYZ and not by the OtsAB pathway. Irrespective of their significance for the osmotic response, otsA and treS were upregulated at the transcriptional level after hyperosmotic shock. In vivo , TreS-mediated trehalose synthesis only occurred if maltose was used as the carbon source. In vitro , TreS catalysed the conversion of maltose into trehalose and, conversely, trehalose into maltose. As the reaction seems to be near equilibrium, TreS appears to be important for trehalose degradation rather than synthesis because a 1000-fold excess of trehalose to maltose was detected in the cytoplasm. Also, evidence is given that both the OtsAB and the TreYZ pathways are involved, but not essential, in supplying trehalose for mycolic acid biosynthesis.
For plants, glutathione conjugation is a major pathway to detoxify organic xenobiotic. Glutathione S-conjugates (SG-conjugates) are formed in the cytosol, the in vitro transport over the tonoplast has been described and a final storage in the vacuole has been postulated. We show here that alachlor rapidly accumulates as GS-conjugates in the plant vacuole and that the first step of its degradation, the formation of the respective Tglutamylcysteinyl-S-conjugate, is catalyzed by a vacuolar carboxypeptidase. These results suggest the glutathione conjugate as a transport form but not a storage form of xenobiotic molecules.
Trehalose (␣-D-glucopyranosyl-␣-D-glucopyranoside) isessential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the socalled Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on ␣-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.
Glutathione S-transferases (GST) detoxify many electrophilic xenobiotics, including several volatile organic compounds and pesticides. The GST activity for the conjugation of several xenobiotic substances was isolated from needles of Norway spruce (Picea abies L. Karst.) trees from a forest decline stand in the northern alps. Trees that exhibited different degrees of damage were selected from several stands in an altitude profile. The GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) in crude protein extracts of needles showed a seasonal pattern with highest activity during summer. The GST activity exhibited a strong dependence on the altitude of the stand showing highest activities in trees growing in the valley and lowest activities in trees growing in the summit regions of the mountain. When cytosolic GST from needles of healthy and damaged trees was purified, trees of healthy appearance exhibited three distinct GST isozymes with activities for the conjugation of CDNB and 1,2-dichloro-4-nitrobenzene (DCNB), whereas severely defoliated trees exhibited four GSTs with additional activity for the conjugation of ethacrynic acid. The main GST isozymes catalyzing the conjugation of CDNB differed in molecular weight, isoelectric point and catalytic properties between damaged and healthy trees.
The synthesis of NAD+ derivatives spin-labeled at either N6 or C8 of the adenine ring is described, in which the carboxamide function of the nicotinamide moiety is replaced by a diazirine ring. Irradiation of these compounds at 3.50 nm generates a carbene which will react with any functional group in its vicinity including hydrocarbons. Both NAD+ derivatives form tight ternary complexes with lactate dehydrogenase and were covalently incorporated into this enzyme. They may be employed for ESR studies when non-covalent interactions are too weak for motionally restricted species to be observed.
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