The minimal genome of the mollicute Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia, encodes a limited repertoire of antioxidant enzymes that include a single and atypical peroxiredoxin (MhPrx), whose evolution and function were studied here. MhPrx has only one catalytic cysteine, in contrast with some of its possible ancestors (2-Cys peroxiredoxins), which have two. Although it is more similar to 2-Cys orthologs, MhPrx can still function with a single peroxidatic cysteine (Cys), using non-thiolic electron donors to reduce it. Therefore, MhPrx could be a representative of a possible group of 2-Cys peroxiredoxins, which have lost the resolving cysteine (Cys) residue without losing their catalytic properties. To further investigate MhPrx evolution, we performed a comprehensive phylogenetic analysis in the context of several bacterial families, including Prxs belonging to Tpx and AhpE families, shedding light on the evolutionary history of Mycoplasmataceae Prxs and giving support to the hypothesis of a relatively recent loss of the Cys within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative Cys caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the Cys of a 2-Cys orthologue, as in the MhPrx supposed ancestral form, was compatible with enzyme activity. Overall, our phylogenetic and mutational studies support that MhPrx recently diverged from a 2-Cys Prx ancestor and pave the way for future studies addressing structural, functional, and evolutive aspects of peroxiredoxin subfamilies in Mollicutes and other bacteria.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in M. hyopneumoniae, important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identified M. hyopneumoniae genes coding for proteins possibly involved with suppression of ROS-mediated damage, one (tpx) coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in Escherichia coli to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing of M. hyopneumoniae extracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection.