Although it plays a central role in the defense of pathogens against oxidative damage, the system formed by the proteins Ohr, an antioxidant enzyme, and OhrR, its transcription factor, has been neglected. This is the opinion of Professor Luis Netto, from the Instituto de Biociências at Universidade de São Paulo (USP) and a member of the RIDC Redoxoma. He has studied Ohr and OhrR proteins for over 20 years and just published an extensive review article analyzing aspects of the structure, catalysis, phylogeny, regulation, and physiological roles of the Ohr/OhrR system. The article was published in the journal Free Radical Biology and Medicine and aims to systematize the information that is dispersed throughout the literature.

Ohr is very efficient in reducing organic hydroperoxides derived from fatty acids and peroxynitrite, two important oxidants in host-pathogen interactions. In addition, studies with genetically modified bacteria indicated the involvement of Ohr or its transcription factor OhrR in virulence. Therefore, the Ohr-OhrR system plays important roles at the interface between pathogens such as Xylella fastidiosa, Pseudomonas aeruginosa, Chromobacterium violaceum, and Bacillus cereus and hosts such as mammals and vascular plants. However, it should be noted that the mechanisms involved in the interactions between pathogens and hosts are complex and the roles played by Ohr and OhrR in these processes vary greatly among different bacteria.

As they are not found in vertebrate animals or vascular plants and have unique structural characteristics, Ohr and OhrR proteins may be targets for the development of new antibiotics, which is very relevant, considering the alarming phenomenon of multidrug-resistant bacteria. In addition, these Ohrs perform other physiological functions, such as protecting symbiotic nitrogen-fixing bacteria from oxidative stress.

Thus, the little attention given to Ohrs is somewhat surprising and, according to Netto, could be explained by historical reasons. The most known antioxidant enzymes were discovered a long time ago: catalase in 1937; SODs in 1969; glutathione peroxidases in 1973; and peroxiredoxins in the early 1990s.

The first Ohr, in turn, was initially described in 1998, as a protein involved in the response of bacteria to stress induced by organic hydroperoxides, and in 2000 Brazilian scientists identified the gene that encodes this protein in the genome of Xylella fastidiosa. This bacterium causes a disease called citrus variegated chlorosis (CVC) that causes great damage to agriculture in the State of São Paulo and was the first phytopathogen to have its genome sequenced. “When Ohr appeared in the Xylella genome, the biochemical activity of this protein was not known. As I had already worked with peroxiredoxins, I saw that Ohr had two very conserved cysteines. That’s when the idea came up: maybe it’s a peroxidase”, says Netto, who was part of the group responsible for the sequencing the genome of this bacterium. The group’s first work on Ohr was published in 2003, in the Journal of Biological Chemistry (JBC), and demonstrated that Ohr is indeed a thiol-dependent peroxidase.

The researcher emphasizes, however, that Ohr is not a peroxiredoxin. “Ohr has very different properties, such as the primary sequence, the structure, the reductant and oxidant specificity, and the dynamics throughout the catalytic cycle”. Peroxiredoxins (Prx), which are also the focus of study by Netto’s group, are antioxidant thiol-proteins considered cellular sensors of hydrogen peroxide.

In 2020, the group elucidated six crystallographic structures of the Ohr protein from the opportunistic pathogen Chromobacterium violaceum, including the structure of the complex between Ohr and its biological substrate, dihydrolipoamide (DHL). The researchers identified intermediates in the enzyme’s catalytic cycle and reinforced evidence that Ohr and Prx belong to different classes of proteins. Furthermore, they showed that Ohr has a hydrophobic collar at its active site, an unique structural feature that explains the enzyme’s specificity for organic peroxides.

Antioxidant family

Ohrs take part of one of three subgroups that make up the family of proteins called Ohr/OsmC, which also includes OsmCs (osmotic shock-induced protein) and Ohr-like proteins. Proteins from the three subgroups (Ohr, OsmC and Ohr-like) have two highly conserved cysteine residues. Ohrs and OsmCs have peroxidase activities based on a highly reactive cysteine, while the Ohr-like group is more heterogeneous and in some cases without any described biochemical activity.

Proteins belonging to the Ohr/OsmC family were initially considered to be present exclusively in bacteria. However, in 2017, Netto’s group analyzed public databases and identified genes from the Ohr/OsmC family in eukaryotes and archaea. Furthermore, the distribution of Ohr/OsmC proteins among different phylogenetic groups is complex and does not show a clear evolutionary relationship, suggesting that the genes were spread through lateral transfer. In fact, the number of genes encoding members of each of the three subgroups varies greatly between different microorganisms.

It should be noted that proteins of the Ohr/OsmC family were found in extremophilic archaea, fungi, and non-vascular plants. By the way, Ohr is present in the bacterium Mycoplasma genitalium, which is the smallest independently living bacterium. Thus, its genome is considered to contain the genes essential to life. On the other hand, the Mycoplasma genitalium genome lacks genes for more popular antioxidants such as SOD and catalase.

Inflammation

The inflammatory response is a host strategy to fight pathogenic microorganisms that involves the production of various oxidants. Therefore, if persistent inflammation takes time to resolve it can cause harm to the host. Fatty acid hydroperoxides are oxidants that can also act as mediators in both inflammatory and anti-inflammatory processes. “The signaling involved in inflammation and the resolution of inflammation is quite complex, involving different hydroperoxides of fatty acids, such as those derived from arachidonic acid”, says Netto.

The hypothesis raised by the researchers is that Ohr may be involved in some way in the virulence of pathogens via the control of lipid hydroperoxides levels. “I believe that Ohr could be involved in the resolution (ending) of the inflammatory process. Counter-intuitively, if the bacterium has a lot of antioxidants (in this case, Ohr), it ends up being bad for it and good for the host, among other reasons, by facilitating the recruitment of phagocytic cells. But this is still a speculative hypothesis that requires experimental evidence”, says the researcher.

OhrR is the protein that controls the transcription of the Ohr gene. For the antioxidant enzyme to be produced, the gene that encodes it needs to be transcribed into the corresponding messenger RNA. When OhrR is in the reduced state, it binds to DNA and prevents transcription. When the cell is under oxidative stress and is exposed to organic hydroperoxides, OhrR undergoes a conformational change and releases DNA, which is then transcribed and translated into the Ohr protein.

According to Netto, “this is beautiful from the Redox perspective: the transcription factor itself is regulated by a redox process and it will induce or repress the expression of a protein that has properties that interfere with the metabolism of organic hydroperoxides”.

Several RIDC Redoxoma groups are involved in studies on lipid hydroperoxides, such as those led by researchers Sayuri Miyamoto and Paolo Di Mascio, as well as in kinetic studies involving Ohr, such as the groups of Ohara Augusto and Daniela Truzzi, and also in structural studies, such as Marcos Antonio de Oliveira’s group.

It is also worth mentioning the participation of undergraduate students Lene Clara M. Santos and Rebeca Bandeira Candia; graduate students José Renato Cussiol, currently at Unifesp, Renato Domingos, currently at Bioline GmbH, Germany and Thiago Alegria, currently a technician at the IB-USP; and post-doctoral fellows Diogo Meireles, currently UENF - Darcy Ribeiro - R, Erika Piccirillo, currently at Eurofarma, and José da Silva Neto, currently at FMRP-USP.

In 2021, a group of Chinese researchers identified the first inhibitor against Ohr from Acinetobacter baumannii ATCC19606. The treatment of this bacterium with this Ohr inhibitor potentiated the antibacterial activity of antibiotics such as kanamycin and gentamicin. The Chinese group contacted Dr. Netto for establishing a collaboration to identify molecules with more potent microbicidal activity.

The article Ohr – OhrR, a neglected and highly efficient antioxidant system: Structure, catalysis, phylogeny, regulation, and physiological roles, by Diogo A. Meireles, José F. da Silva Neto, Renato M. Domingos, Thiago G. P. Alegria, Lene Clara M. Santos, and Luis Eduardo S. Netto can be accessed here.