Redoxoma

CEPID Redoxoma

RIDC Redoxoma


Study details the mechanism of action of a bacterial antioxidant protein

Ohr (Organic hydroperoxide resistance protein) is a potential drug target
PorBy Maria Celia Wider
• CEPIDRIDC Redoxoma
03/06/2020
São Paulo, Braszil

Researchers led by Professor Luis Eduardo Soares Netto, from Instituto de Biociências at Universidade de São Paulo (USP) and a member of RIDC Redoxoma, described six crystallographic structures of the Ohr enzyme from the opportunistic pathogen Chromobacterium violaceum, including the structure of the complex between Ohr and its biological substrate, the dihydrolipoamide (DHL), and analyzed the structural changes along the catalytic cycle of the enzyme. Ohr is an antioxidant enzyme present mainly in bacteria and fungi, some of them are pathogenic, protecting them from oxidative stress. The study, published in the ACS Catalysis journal, was carried out during the doctorate of Renato Mateus Domingos, the first author and corresponding co-author of the article, and may facilitate the development of an enzyme inhibitor.

“We used several approaches, such as crystallography, molecular dynamics, and kinetics to describe our model, with a focus on the reduction phase in the Ohr catalytic cycle, which was the most elusive one. The structure of the enzyme in complex with its substrate provided structural ground for the theoretical simulations. Knowing the enzymatic mechanism makes it easier - or less difficult - to design Ohr inhibitors”, said Luis Netto. As there are no similar enzymes in mammals or plants, Ohr is an attractive new target for drug development. Identifying inhibitors of this enzyme would also be important for agriculture, as it has already been found in pathogens - bacteria and fungi - that attack plants.

The relationships between pathogens and hosts are complex and have been modulated during the evolutionary process. When invaded by pathogenic microorganisms, plants and animals trigger an inflammatory response, with the generation of oxidants, including hydroperoxides derived from fatty acids. Bacteria, in turn, “counterattack” with an arsenal of enzymes to break down these oxidants. Ohr is one of those enzymes that plays a central role in the bacterial response to oxidants, such as fatty acid hydroperoxides and peroxynitrite. For that reason, Ohr has been associated with the virulence of some bacteria, such as Pseudomonas aeruginosa, Chromobacterium violaceum, and Pseudomonas aeruginosa.

Superbugs, or multi-drug resistant microorganisms (MDR), are a worldwide threat to public health. The World Health Organization has been warning that we may be heading towards a post-antibiotic era, in which common infections and minor injuries can kill again. Therefore, the identification of new targets for the development of antimicrobial treatments is a global urgency.

Scheme shows the Ohr, its biological reductant (DHL), and the reduction phase of the catalytic cycle
Scheme shows the Ohr, its biological reductant (DHL), and the reduction phase of the catalytic cycle — Luis E. S. Netto

Catalytic mechanism

“Ohr is an enzyme capable of accelerating the peroxide decomposition reaction by a factor of a million times compared to the non-catalyzed reaction. To understand this high efficiency, it was very important to know intermediates of the catalysis, and the structures that Renato described allowed us to achieve this goal”, Luis Netto said.

Crystallographic structures are like photographs, showing a static moment, explains the researcher. But proteins, to perform their function, are in motion, like in a film. To visualize the catalytic cycle of an enzyme, many photos are needed - as in that old animation processes in which a sequence of drawings passed at speed gave the impression of movement.

To determine the structures described in the study, several steps were necessary, starting with the cloning of the Ohr gene. Next, the researchers obtained large amounts of the protein using the bacterium E. coli, which received the gene that contains the molecular instructions to generate the Ohr protein. After that, it was necessary to purify Ohr, isolating it from the other E. coli proteins. As the purified protein was in solution, the next step was to form the crystals. Like the salt used for cooking, protein molecules also can organize themselves in solid state, which are the crystals. These crystals were taken to Stanford University’s synchrotron - Stanford Synchrotron Radiation Light source (SSRL) and to the Laboratório Nacional de Luz Sincrotron em Campinas (LNLS)-Brazil, where they were exposed to X-ray, which produced diffraction patterns, providing information to determine the Ohr structures, including the one with DHL bound to the enzyme. Through mathematical calculations, the researchers determined the electronic densities, to which chemical groups were attributed, and comprise the three dimensional structures of the enzyme. The validation of these structures was done in collaboration with the groups of Raphael D. Teixeira / Shaker Chuck Farah, from the Instituto de Química/USP, and Plínio S. Vieira / Mario Murakami, from the National Biosciences Laboratory (LNBio) of the National Research Center for Energy and Materials (CNPEM).

The researchers also compared the structures they obtained with the 10 other Ohr structures available in the PDB (Protein Data Bank), confirming the results and the proposed catalytic cycle. “The more structures of the same protein are available in the database, the more consistently we can propose models. With our work, we greatly increased the amount of Ohr structures available. This allow us to perform analyses with more confidence” Netto said.

To understand Ohr’s catalytic mechanism based on its structures, the researchers used several molecular modeling approaches, such as classical mechanics (MM), steered molecular dynamics (SMD), and hybrid quantum mechanics (QM-MM), in collaboration with researcher Dario A. Estrin, from the Universidad de Buenos Aires. These simulations together with experimental tests employing site-directed mutagenesis in Ohr revealed that the substrates and products assist conformational changes and thereby accelerate enzyme turnover. According to the researchers, the results support the concept that DHL is an Ohr biological reductant. Also, they expand the concept of substrate and product assisted catalysis to a new class of enzymes, Ohr proteins.

The article Substrate and product-assisted catalysis: molecular aspects behind structural switches along Organic Hydroperoxide Resistance Protein catalytic cycle, by Renato M. Domingos, Raphael D. Teixeira, Ari Zeida, William A. Agudelo, Thiago G.P. Alegria, Jose F. da Silva Neto, Plínio S. Vieira, Mario T. Murakami, Chuck S. Farah, Dario A. Estrin, and Luis E.S. Netto, can be accessed here.