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Xylella fastidiosa is a gram-negative bacterium, which colonizes the xylem from economically important plants, being responsible for several diseases such as Pierce disease (PD) in gravepines and citrus variegated clorosis (CVC). Plants, when infected by pathogens, are able to defend themselves through several mechanisms which include the generation of reactive oxygen species (ROS). Lipid hydroperoxides can be generated from the attack of ROS to the bacterial membrane or by the action of lipoxygenases. The alkyl hydroperoxide reductase system (AhpR) was initially characterized as the main responsible for the detoxification of organic hydroperoxides in bacteria. Recently, it was also characterized another gene in many pathogenic bacteria, whose deletion renders cells susceptibility to organic hydroperoxide treatments but not by H2O2 or by superoxide generators (Mongkolsuk et al., 1998 and Ochsner et al., 2001). For this reason, it was named ohr (organic hydroperoxide resistance gene). The goal of this work was to functionally characterize Ohr, the product of ohr gene from Xylella fastidiosa. Initially, we demonstrated that Ohr possesses Cys-based thiol-dependent peroxidase activity. Later, we showed that Ohr possesses a unique alpha/beta fold not observed in the structures of other thiol peroxidases such as peroxiredoxins and glutathione peroxidases. Analyses of Ohr active site showed that its likely substrates are elongated and hydrophobic molecules. Furthermore, we showed that lipoylated enzymes, classically related with the intermediary metabolism, interacts physically and functionally with Ohr while classical thiol-dependent pathways, such as thioredoxin and glutathione, failed to support Ohr activity. This finding represents the first evidence of a peroxidase that is directly reduced by lipoyl groups of enzymes. Also, we obtained evidences indicating that Ohr acts in the detoxification of peroxides derived from unsaturated fatty acids. In fact, steady-state kinetics using bi-substrate analysis showed that Ohr decomposes organic peroxides with high efficiency (kcat/KM ~ 10 6 M.s) through a pingpong mechanism, at least ten thousand times more efficiently than hydrogen peroxide (H2O2). All these results together shows that Ohr is central in the response of bacteria to the stress induced by organic hydroperoxides but not by H2O2 and defines a new class of antioxidant enzymes with unique properties such as lipoyl-dependent peroxidase activity. Another goal of this work was to study the regulation of ohr expression in Xylella fastidiosa. ohr expression is regulated in most bacteria by a repressor protein named OhrR (Sukchawalit et al., 2001) but, in some bacteria, ohr expression is positively regulated by an alternative sigma factor (σ) with extracitoplasmatic function (Gourion et al., 2008). Our results showed that ohr from X. fastidiosa was not under the control of none of these regulators, probably being constitutively expressed. Through northern blot analysis, we did not observed any changes in ohr levels in cells submitted to oxidative or ethanolic stress. These results, indicates that ohr expression probably differs from that previously described on literature for other bacteria.

Caracterização funcional de uma nova proteína antioxidante: Ohr (Organic Hydroperoxide Resistance Protein). Vias de redução e expressão em Xylella fastidiosa