Abstract
ohr (organic hydroperoxide resistance gene) is present in several species of bacteria, and its deletion renders cells specifically sensitive to organic peroxides. The goal of this work was to determine the biochemical function of Ohr from Xylella fastidiosa. All of the Ohr homologues possess two cysteine residues, one of them located in a VCP motif, which is also present in all of the proteins from the peroxiredoxin family. Therefore, we have investigated whether Ohr possesses thiol-dependent peroxidase activity. The ohr gene from X. fastidiosa was expressed in Escherichia coli, and the recombinant Ohr decomposed hydroperoxides in a dithiothreitol-dependent manner. Ohr was about twenty times more efficient to remove organic hydroperoxides than to remove H(2)O(2). This result is consistent with the organic hydroperoxide sensitivity of Delta ohr strains. The dependence of Ohr on thiol compounds was ascertained by glutamine synthetase protection assays. Approximately two thiol equivalents were consumed per peroxide removed indicating that Ohr catalyzes the following reaction: 2RSH + ROOH --> RSSR + ROH + H(2)O. Pretreatment of Ohr with N-ethyl maleimide and substitution of cysteine residues by serines inhibited this peroxidase activity indicating that both of the Ohr cysteines are important to the decomposition of peroxides. C125S still had a residual enzymatic activity indicating that Cys-61 is directly involved in peroxide removal. Monothiol compounds do not support the peroxidase activity of Ohr as well as thioredoxin from Saccharomyces cerevisiae and from Spirulina. Interestingly, dithiothreitol and dyhydrolipoic acid, which possess two sulfhydryl groups, do support the peroxidase activity of Ohr. Taken together our results unequivocally demonstrated that Ohr is a thiol-dependent peroxidase.
Highlights
The infection of both plants and animals induces a defense response that results in an oxidative burst with the increased generation of ROS1 (1)
The ohr gene is present in a single copy, but in some cases such as B. subtilis, Mesorhizobium loti, and Ralstonia solanacearum two copies of ohr are present (12, 22, 23)
A blastp analysis on the X. fastidiosa genome using the tools available at the site aeg.lbi.ic.unicamp.br/xf/ detected only one copy of ohr in this bacteria located between coordinates 1,742,868 and 1,743,299 with 432 nucleotides
Summary
The infection of both plants and animals induces a defense response that results in an oxidative burst with the increased generation of ROS1 (1). The alkyl hydroperoxide reductase (AhpR) is frequently considered the main enzyme responsible for the conversion of organic peroxides to the corresponding alcohols in bacteria (6, 7). This enzyme comprises two subunits, AhpF and AhpC. Phaseoli because its deletion rendered cells highly sensitive to killing by organic peroxides but not to H2O2 or superoxide generators (10). It was named organic hydroperoxide resistance (ohr) gene. Despite the suggestions that Ohr might directly detoxify organic hydroperoxides, it was not possible to rule out the possibility that Ohr is involved in other processes such as the transport of organic molecules (10) or in yet undefined signal-
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
