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

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.

Slx4 scaffolding in homologous recombination and checkpoint control: lessons from yeast.

Homologous recombination-mediated DNA repair is essential for maintaining genome integrity. It is a multi-step process that involves resection of DNA ends, strand invasion, DNA synthesis and/or DNA end ligation, and finally, the processing of recombination intermediates such as Holliday junctions or other joint molecules. Over the last 15 years, it has been established that the Slx4 protein plays key roles in the processing of recombination intermediates, functioning as a scaffold to coordinate the action of structure-specific endonucleases. Recent work in budding yeast has uncovered unexpected roles for Slx4 in the initial step of DNA-end resection and in the modulation of DNA damage checkpoint signaling. Here we review these latest findings and discuss the emerging role of yeast Slx4 as an important coordinator of DNA damage signaling responses and a regulator of multiple steps in homologous recombination-mediated repair.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite.

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108 M-1⋅s-1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M-1⋅s-1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.

Termination of Replication Stress Signaling via Concerted Action of the Slx4 Scaffold and the PP4 Phosphatase.

In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.

Dampening DNA damage checkpoint signalling via coordinated BRCT domain interactions.

In response to DNA damage, checkpoint signalling protects genome integrity at the cost of repressing cell cycle progression and DNA replication. Mechanisms for checkpoint down-regulation are therefore necessary for proper cellular proliferation. We recently uncovered a phosphatase-independent mechanism for dampening checkpoint signalling, where the checkpoint adaptor Rad9 is counteracted by the repair scaffolds Slx4-Rtt107. Here, we establish the molecular requirements for this new mode of checkpoint regulation. We engineered a minimal multi-BRCT-domain (MBD) module that recapitulates the action of Slx4-Rtt107 in checkpoint down-regulation. MBD mimics the damage-induced Dpb11-Slx4-Rtt107 complex by synergistically interacting with lesion-specific phospho-sites in Ddc1 and H2A. We propose that efficient recruitment of Dpb11-Slx4-Rtt107 or MBD via a cooperative 'two-site-docking' mechanism displaces Rad9. MBD also interacts with the Mus81 nuclease following checkpoint dampening, suggesting a spatio-temporal coordination of checkpoint signalling and DNA repair via a combinatorial mode of BRCT-domains interactions.

Ohr (organic hydroperoxide resistance protein) possesses a previously undescribed activity, lipoyl-dependent peroxidase.

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa.

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.