Inhibitory effect of metals on animal and plant glutathione transferases.

Glutathione transferases (GSTs) represent a widespread enzyme superfamily in eukaryotes and prokaryotes catalyzing different reactions with endogenous and xenobiotic substrates such as organic pollutants. The latter are often found together with metal contamination in the environment. Besides performing of essential functions, GSTs protect cells by conjugation of glutathione with various reactive electrophiles. The interference of toxic metals with this functionality of GSTs may have unpredictable toxicological consequences for the organisms. In this review results from the recent literature are summarized and discussed describing the ability of metals to inhibit intracellular detoxification processes in animals and plants.

Tissue-specific metabolism of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss): a comparison between the liver and immune organs.

Polycyclic aromatic hydrocarbons (PAHs) are immunotoxicants in fish. In mammals, phase I metabolites are believed to be critically involved in the immunotoxicity of PAHs. This mechanism has been suggested for fish as well. The present study investigates the capacity of immune organs (head kidney, spleen) of rainbow trout, Oncorhynchus mykiss, to metabolize the prototypic PAH, benzo[a]pyrene (BaP). To this end, we analyzed 1) the induction of enzymatic capacity measured as 7-ethoxyresorufin-O-deethylase (EROD) activity in immune organs compared with liver, 2) the organ profiles of BaP metabolites generated in vivo, and 3) rates of microsomal BaP metabolite production in vitro. All measurements were done for control fish and for fish treated with an intraperitoneal injection of 15 mg BaP/kg body weight. In exposed trout, the liver, head kidney, and spleen contained similar levels of BaP, whereas EROD induction differed significantly between the organs, with liver showing the highest induction factor (132.8×), followed by head kidney (38.4×) and spleen (1.4×). Likewise, rates of microsomal metabolite formation experienced the highest induction in the liver of BaP-exposed trout, followed by the head kidney and spleen. Microsomes from control fish displayed tissue-specific differences in metabolite production. In contrast, in BaP-exposed trout, microsomes of all organs produced the potentially immunotoxic BaP-7,8-dihydrodiol as the main metabolite. The findings from this study show that PAHs, like BaP, are distributed into immune organs of fish and provide the first evidence that immune organs possess inducible PAH metabolism leading to in situ production of potentially immunotoxic PAH metabolites.

Regulation of sulfate assimilation in Physcomitrella patens: mosses are different!

Sulfur is an essential nutrient, taken up as sulfate from soil, reduced and incorporated into bioorganic compounds in plant cells. The pathway of sulfate assimilation is highly regulated in a demand-driven manner in seed plants. To test the evolutionary conservation of the regulatory mechanisms, we analyzed regulation of the pathway in the model for basal plants, the moss Physcomitrella patens. While in Arabidopsis the key enzyme of sulfate assimilation, adenosine 5'-phosphosulfate reductase (APR), is feedback repressed by thiols and induced by reduced levels of glutathione, in P. patens such regulation does not occur. The control of the pathway was not moved to other components as these conditions affected neither mRNA accumulation of other genes of sulfate assimilation nor sulfate uptake. Other treatments known to regulate APR, O-acetylserine, cadmium and sulfur deficiency affected APR transcript levels, but not enzyme activity. It appears that the sulfate assimilation pathway in P. patens is much more robust than in seed plants. Thus, the regulatory networks controlling the pathway have probably evolved only later in the evolution of the seed plants after separation of the bryophytes.

Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development.

A key step in sulfate assimilation into cysteine is the reduction of sulfite to sulfide by sulfite reductase (SiR). This enzyme is encoded by three genes in the moss Physcomitrella patens. To obtain a first insight into the roles of the individual isoforms, we deleted the gene encoding the SiR1 isoform in P. patens by homologous recombination and subsequently analysed the DeltaSiR1 mutants. While DeltaSiR1 mutants showed no obvious alteration in sulfur metabolism, their regeneration from protoplasts and their ability to produce mature spores was significantly affected, highlighting an unexpected link between moss sulfate assimilation and development, that is yet to be characterized.

Adenylyl cyclase Rv0386 from Mycobacterium tuberculosis H37Rv uses a novel mode for substrate selection.

Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA-ATPase and a helix-turn-helix DNA-binding domain. In Rv0386, the standard substrate, adenine-defining lysine-aspartate couple is replaced by glutamine-asparagine. The recombinant adenylyl cyclase domain was active with a V(max) of 8 nmol cAMP.mg(-1).min(-1). Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side-activity with GTP as a substrate. Mutation of the glutamine-asparagine pair either to alanine residues or to the canonical lysine-aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two non-canonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.