Substrate and Product-Assisted Catalysis: Molecular Aspects behind Structural Switches along Organic Hydroperoxide Resistance Protein Catalytic Cycle

TEIXEIRA, RAPHAEL D.; ALEGRIA, THIAGO G. P.; MURAKAMI, MARIO T.; NETTO, LUIS E. S.; ZEIDA, ARI; DA SILVA NETO, JOSÉ F.; FARAH, CHUCK S.; DOMINGOS, RENATO M.; AGUDELO, WILLIAM A.; VIEIRA, PLÍNIO S.; ESTRIN, DARIO A.

ACS Catalysis

American Chemical Society

Año: 2020 vol. 10 p. 6587 - 6602

2155-5435

Bacteria contain a large repertoire of enzymes to decompose oxidants, such as hydroperoxides. Among them, organic hydroperoxide resistance (Ohr) proteins play central roles in the bacterial response to fatty acid peroxides and peroxynitrite (Alegria et al. Ohr Plays a Central Role in BacterialResponses against Fatty Acid Hydroperoxides and Peroxynitrite (Proc. Natl. Acad. Sci. USA 2017, 114, E132) and present distinct structural and biochemical features in comparison with mammalianCys-based peroxidases. The molecular events associated with the high reactivity of Ohr enzymes toward hydroperoxides and its reducibility by lipoylated proteins (or dihydrolipoamide) are still elusive. Here, we report six crystallographic structures of two Ohr paralogs from Chromobacterium violaceum, including the complex with dihydrolipoamide. Comparison of these six structures with the other few Ohr structures available in public databases revealed conserved features in the active site, such as a hydrophobic collar. Together with classical, hybrid quantum-classical molecular dynamics simulations and point mutation analyses, we show that Ohr undergoes several structural switches to allow an energetically accessible movement of the loop containing the catalytic Arg, which is stabilized in the closed state when the catalytic Cys is reduced. The structure of Ohr in complex with its substrate (dihydrolipoamide) together with molecular simulations allowed us tocharacterize the reductive half of the catalytic pathway in detail. Notably, dihydrolipoamide favors Arg-loop closure, thereby assisting enzyme turnover. The conserved physicochemical properties of the Ohr active site and the mechanisms revealed here provide relevant information for the identification of inhibitors with therapeutic potential.