Novel class of glutathione transferases from cyanobacteria exhibit high catalytic activities towards naturally occurring isothiocyanates

In the present paper, we report a novel class of GSTs (glutathione transferases), called the Chi class, originating from cyanobacteria and with properties not observed previously in prokaryotic enzymes. GSTs constitute a widespread multifunctional group of proteins, of which mammalian enzymes are the best characterized. Although GSTs have their origin in prokaryotes, few bacterial representatives have been characterized in detail, and the catalytic activities and substrate specificities observed have generally been very modest. The few well-studied bacterial GSTs have largely unknown physiological functions. Genome databases reveal that cyanobacteria have an extensive arsenal of glutathione-associated proteins. We have studied two cyanobacterial GSTs which are the first examples of bacterial enzymes that are as catalytically efficient as the best mammalian enzymes. GSTs from the thermophile Thermosynechococcus elongatus BP-1 and from Synechococcus elongatus PCC 6301 were found to catalyse the conjugation of naturally occurring plant-derived isothiocyanates to glutathione at high rates. The cyanobacterial GSTs studied are smaller than previously described members of this enzyme family, but display many of the typical structural features that are characteristics of GSTs. They are also active towards several classical substrates, but at the same moderate rates that have been observed for other GSTs derived from prokaryotes. The cloning, expression and characterization of two cyanobacterial GSTs are described. The possible significance of the observed catalytic properties is discussed in the context of physiological relevance and GST evolution

Dietary exposure of Daphnia to microcystins: No in vivo relevance of biotransformation

Anthropogenic nutrient input into lakes has contributed to the increased frequency of toxic cyanobacterial blooms. Daphnia populations have been shown to be locally adapted to toxic cyanobacteria and are able to suppress bloom formation; little is known about the physiology behind this phenomenon. Microcystin-LR (MCLR) is the most widespread cyanobacterial toxin, and, based on in vitro experiments, it is assumed that the enzyme glutathione-S-transferase (GST) might act as the first step of detoxification in Daphnia by conjugating MCLR with glutathione. In the present study Daphnia magna was fed a diet of 100% Microcystis aeruginosa PCC7806, a cyanobacterial strain that contains MCLR in high amounts (4.8-5.6 fg cell(-1)), in order to test for a possible conjugation of MCLR with GST in Daphnia in vivo. We used high-resolution LCMS to analyze incubation water, cyanobacterial cells and Daphnia tissue for the presence of MCLR conjugation products as well as unconjugated MCLR. Newly formed conjugation products were detected neither in Daphnia tissue nor in the incubation water. Moreover, the presence of Daphnia led to a decrease in unconjugated MCLR in the cyanobacterial cell fraction due to grazing, in comparison to a control without daphnids, which was well reflected by a similar increase of MCLR in the respective incubation water. As a consequence, the MCLR content did not change due to Daphnia presence within the entire experimental setup. In summary, MCLR ingestion by Daphnia led neither to the formation of conjugation products, nor to a decrease of unconjugated MCLR. GST-mediated conjugation thus seems to be of minor relevance for microcystin (MC) tolerance in Daphnia in vivo. This finding is supported by the fact that GST activity in Daphnia feeding on the MC-containing wildtype or a MC-free mutant of M. aeruginosa PCC7806 revealed an identical increase of specific activity in comparison to a cyanobacteria-free diet. Therefore, the frequently observed induction of GST activity upon exposure to toxic cyanobacteria is not a specific MC effect but a general cyanobacterial effect. This suggests that GST in Daphnia is involved in an oxidative stress response rather than in the specific detoxification of MCs. Furthermore, our results indicate the presence of an efficient transport mechanism which efficiently removes unconjugated MCLR from the Daphnia tissue. Further studies are needed to elucidate the nature of this transport mechanism. (C) 2014 Elsevier B.V. All rights reserved