Structural insights on the efficient catalysis of hydroperoxide reduction by Ohr: Crystallographic and molecular dynamics approaches

Abstract

Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from Xylella fastidiosa) and Glu (Glu51 in Ohr from Xylella fastidiosa) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (Cp) toward hydroperoxides. In the closed state, the thiolate of Cp is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from Cp and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from Xylella fastidiosa (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and in silico mutagenesis indicated that the polar interactions of Arg19 with Glu51 and Cp contributed to the stabilization of XfOhr in the closed state. Indeed, Cp oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while Cp remains relatively static.

Catalysis of Peroxide Reduction by Fast Reacting Protein Thiols

“…In particular, polar interactions among these three residues are important to stabilize a “closed” conformation of the reduced protein and to activate the thiolate for the reduction of hydroperoxides. Oxidation to the intramolecular disulfide form triggers changes in the interactions of the conserved Glu and in the dynamics of the conserved Arg which favor the “open” conformation which is thought to facilitate reduction. , …”

“…Oxidation to the intramolecular disulfide form triggers changes in the interactions of the conserved Glu and in the dynamics of the conserved Arg which favor the "open" conformation which is thought to facilitate reduction. 303,311 Other proteins with thiols highly reactive toward hydroperoxides include the bacterial transcription factors OxyR and OhrR that control bacterial transcriptional responses to hydrogen peroxide (rate constant of ∼10 5 M −1 s −1 ) 312,313 and organic hydroperoxides, respectively, 312,314,315 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic pathway enzyme that has been recently proposed to be involved in DNA repair and heme binding and delivery to target proteins, among other functions, 316,317 that reacts with H 2 O 2 with rate constant in the 10 2 −10 3 M −1 s −1 range. 318 An important case of protein inactivation due to Cys oxidation by hydroperoxides are the protein tyrosine phosphatases (PTP), enzymes that catalyze the hydrolysis of phosphotyrosine from specific signal-transducing proteins.…”

The Human Pathogen Paracoccidioides brasiliensis Has a Unique 1-Cys Peroxiredoxin That Localizes Both Intracellularly and at the Cell Surface

“…The fact that PbPrx1 can decompose organic hydroperoxides more efficiently than hydrogen peroxide is not an exclusive characteristic of PbPrx1. Other thiol peroxidases, such as the human PrxV, E. coli thiol peroxidase (EcTpx), and the organic hydroperoxide resistance protein (Ohr) from Xylella fastidiosa are 100–1000-fold more reactive with organic peroxides, as a consequence of a hydrophobic active site microenvironment, which enables interactions with hydrophobic oxidizing substrates (Cussiol et al, 2003 ; Perkins et al, 2014 ; Alegria et al, 2017 ; Piccirillo et al, 2018 ). In this context, our data suggest that the active site microenvironment of PbPrx1 is also highly hydrophobic, suggesting that PbPrx1 may act as a strong scavenger of organic peroxides in P. brasiliensis cells.…”

A small molecule interacts with pMAC-derived hydroperoxide reductase and enhances the activity of aminoglycosides

“…Compared with the wild-type (WT) parent strain A. baumannii ATCC19606, mutations located in plasmid (pMAC)-derived ohrB were identified both in 3M and 5M strains, resulting in the conversion of arginine at the position 15 (Arg15) to His or Cys of OhrB (Table 2). indicated that the corresponding Arg19 in Ohr from Xylella fastidiosa contributed to the stabilization of XfOhr in the closed state, suggesting that the mutations in 3M and 5M probably affect the function of OhrB [13].…”