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 (CysP), 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 (CysR) 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 CysR within this family. Moreover, mutational analyses provided insights into MhPrx function with one, two, or without catalytic cysteines. While removal of the MhPrx putative CysP caused complete activity loss, confirming its catalytic role, the introduction of a second cysteine in a site correspondent to that of the CysR 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.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). T. G. was a recipient of a CNPq PIBITI fellowship. C. E. T. is recipient of a CAPES post-doctoral fellowship. V. G. V. was a recipient of a CAPES post-doctoral fellowship. C. X. M. was a recipient of a CAPES M.Sc. fellowship. J. A. P. is a recipient of a CAPES Ph.D. fellowship. We also thank T. Reinaldo, for the help with the design of megaprimers, and Dr. D. Maturana, for the help with thermophoretic dimerization assays.
Supplementary material 1 Fig. S1 Bacterial Prx evolutionary tree obtained using bayesian inference implemented by the MrBayes software. The posterior probabilities higher or equal to 0.7 are shown. The conserved cysteine residue for each bacterial family is indicated below the family label. Additional cysteine residues found in specific Prx sequences are indicated after the species name. The list of organisms from which Prxs sequences was taken for the global alignments (Fig. S2), their corresponding sequence accession numbers, and cysteine positions are in Supplementary Table S1 (TIFF 15168 kb)
Supplementary material 2 Fig. S2 Multiple alignment of all Prx sequences used for the construction of the phylogenetic tree shown in Supplementary Fig. S1. The positions of all cysteines can be found in Table S1 (TIFF 9573 kb)
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