John Michael Atack

The Campylobacter jejuni Thiol Peroxidases Tpx and Bcp Both Contribute to Aerotolerance and Peroxide-Mediated Stress Resistance but Have Distinct Substrate Specificities

The microaerophilic food-borne pathogen Campylobacter jejuni experiences variable oxygen concentrations during its life cycle, especially during transitions between the external environment and the avian or mammalian gut. Single knockout mutations in either one of two related thiol peroxidase genes, tpx and bcp, resulted in normal microaerobic growth (10% [vol/vol] oxygen) but poorer growth than that of the wild type under high-aeration conditions (21% [vol/vol] oxygen). However, a tpx/bcp double mutant had a severe microaerobic growth defect and did not grow at high aeration in shake flasks. Although the single mutant strains were no more sensitive than the wild-type strains in disc diffusion assays with hydrogen peroxide, organic peroxides, superoxide, or nitrosative stress agents, in all cases the double mutant was hypersensitive. Quantitative cell viability and cellular lipid peroxidation assays indicated some increased sensitivity of the single tpx and bcp mutants to peroxide stress. Protein carbonylation studies revealed that the tpx/bcp double mutant had a higher degree of oxygen- and peroxide-induced oxidative protein damage than did either of the single mutants. An analysis of the peroxidase activity of the purified recombinant enzymes showed that, surprisingly, Tpx reduced only hydrogen peroxide as substrate, whereas Bcp also reduced organic peroxides. Immunoblotting of wild-type cell extracts with Tpx- or Bcp-specific antibodies showed increased abundance of both proteins under high aeration compared to that under microaerobic growth conditions. Taken together, the results suggest that Tpx and Bcp are partially redundant antioxidant enzymes that play an important role in protection of C. jejuni against oxygen-induced oxidative stress.

Oxidative stress in Campylobacter jejuni: responses, resistance and regulation

Campylobacter jejuni is a major food-borne human pathogen that paradoxically is an oxygen-sensitive microaerophile, yet must resist the oxidative stresses encountered both in the host and in the environment. Recent studies suggest that, perhaps surprisingly, C. jejuni contains a wide range of enzymes involved in oxidative stress defense, and this review focuses on the properties and roles of these proteins. Although the mechanisms of gene regulation are still poorly understood in C. jejuni, several regulators of the oxidative stress response have been identified and their properties are discussed here. We suggest that future studies should be directed towards identifying the role of additional and less well characterized components involved in oxidative stress resistance, as well as providing a more complete picture of the underlying sensing and regulatory mechanisms.

Structure, Mechanism and Physiological Roles of Bacterial Cytochrome c Peroxidases.

Cytochrome-c peroxidases (CCPs) are a widespread family of enzymes that catalyse the conversion of hydrogen peroxide (H2O2) to water using haem co-factors. CCPs are found in both eukaryotes and prokaryotes, but the enzymes in each group use a distinct mechanism for catalysis. Eukaryotic CCPs contain a single b-type haem co-factor. Conventional bacterial CCPs (bCCPs) are periplasmic enzymes that contain two covalently bound c-type haems. However, we have identified a sub-group of bCCPs by phylogenetic analysis that contains three haem-binding motifs. Although the structure and mechanism of several bacterial di-haem CCPs has been studied in detail and is well understood, the physiological role of these enzymes is often much less clear, especially in comparison to other peroxidatic enzymes such as catalase and alkyl-hydroperoxide reductase. In this review, the structure, mechanism and possible roles of bCCPs are examined in the context of their periplasmic location, the regulation of their synthesis by oxygen and their particular function in pathogens.

Unpairing and gating: sequence-independent substrate recognition by FEN superfamily nucleases.

Structure-specific 5-nucleases form a superfamily of evolutionarily conserved phosphodiesterases that catalyse a precise incision of a diverse range of DNA and RNA substrates in a sequence-independent manner. Superfamily members, such as flap endonucleases, exonuclease 1, DNA repair protein XPG, endonuclease GEN1 and the 5-3-exoribonucleases, play key roles in many cellular processes such as DNA replication and repair, recombination, transcription, RNA turnover and RNA interference. In this review, we discuss recent results that highlight the conserved architectures and active sites of the structure-specific 5-nucleases. Despite substrate diversity, a common gating mechanism for sequence-independent substrate recognition and incision emerges, whereby double nucleotide unpairing of substrates is required to access the active site.

Substrate recognition and catalysis by flap endonucleases and related enzymes

FENs (flap endonucleases) and related FEN-like enzymes [EXO-1 (exonuclease-1), GEN-1 (gap endonuclease 1) and XPG (xeroderma pigmentosum complementation group G)] are a family of bivalent-metal-ion-dependent nucleases that catalyse structure-specific hydrolysis of DNA duplex-containing nucleic acid structures during DNA replication, repair and recombination. In the case of FENs, the ability to catalyse reactions on a variety of substrates has been rationalized as a result of combined functional and structural studies. Analyses of FENs also exemplify controversies regarding the two-metal-ion mechanism. However, kinetic studies of T5FEN (bacteriophage T5 FEN) reveal that a two-metal-ion-like mechanism for chemical catalysis is plausible. Consideration of the metallobiochemistry and the positioning of substrate in metal-free structures has led to the proposal that the duplex termini of substrates are unpaired in the catalytically active form and that FENs and related enzymes may recognize breathing duplex termini within more complex structures. An outstanding issue in FEN catalysis is the role played by the intermediate (I) domain arch or clamp. It has been proposed that FENs thread the 5-portion of their substrates through this arch, which is wide enough to accommodate single-stranded, but not double-stranded, DNA. However, FENs exhibit gap endonuclease activity acting upon substrates that have a region of 5-duplex. Moreover, the action of other FEN family members such as GEN-1, proposed to target Holliday junctions without termini, appears incompatible with a threading mechanism. An alterative is that the I domain is used as a clamp. A future challenge is to clarify the role of this domain in FENs and related enzymes.

Flap endonucleases pass 5′-flaps through a flexible arch using a disorder-thread-order mechanism to confer specificity for free 5′-ends

Flap endonucleases (FENs), essential for DNA replication and repair, recognize and remove RNA or DNA 5′-flaps. Related to FEN specificity for substrates with free 5′-ends, but controversial, is the role of the helical arch observed in varying conformations in substrate-free FEN structures. Conflicting models suggest either 5′-flaps thread through the arch, which when structured can only accommodate single-stranded (ss) DNA, or the arch acts as a clamp. Here we show that free 5′-termini are selected using a disorder-thread-order mechanism. Adding short duplexes to 5′-flaps or 3′-streptavidin does not markedly impair the FEN reaction. In contrast, reactions of 5′-streptavidin substrates are drastically slowed. However, when added to premixed FEN and 5′-biotinylated substrate, streptavidin is not inhibitory and complexes persist after challenge with unlabelled competitor substrate, regardless of flap length or the presence of a short duplex. Cross-linked flap duplexes that cannot thread through the structured arch react at modestly reduced rate, ruling out mechanisms involving resolution of secondary structure. Combined results explain how FEN avoids cutting template DNA between Okazaki fragments and link local FEN folding to catalysis and specificity: the arch is disordered when flaps are threaded to confer specificity for free 5′-ends, with subsequent ordering of the arch to catalyze hydrolysis.

Contribution of the stereospecific methionine sulphoxide reductases MsrA and MsrB to oxidative and nitrosative stress resistance in the food-borne pathogen Campylobacter jejuni

The microaerophilic food-borne pathogen Campylobacter jejuni is exposed to highly variable oxygen concentrations during its life cycle and employs a variety of protection mechanisms to resist oxidative stress. However, not all of the enzymes that mediate such protection have yet been identified. Two genes in strain NCTC 11168, Cj0637c and Cj1112c, are predicted to encode unrelated methionine sulphoxide reductases, which may repair oxidized methionine residues in proteins and thus contribute to oxidative stress defence. Cj0637 and Cj1112 were overexpressed, purified and shown by a coupled thioredoxin-thioredoxin reductase-NADPH assay to catalyse the stereospecific reduction of the S and R diastereoisomers, respectively, of the model compound methyl p-tolyl sulphoxide. Cj0637 is thus identified as MsrA and Cj1112 as MsrB. The contribution of these enzymes to oxidative and nitrosative stress resistance in C. jejuni was assessed by phenotypic analysis of a set of isogenic msrA, msrB and msrA/B insertion mutants. As RT-PCR data suggested a polar effect on Cj1111c in the msrB mutant, an msrB/msrB(+) merodiploid complementation strain was also constructed. The msrA/B strain was severely growth inhibited under standard microaerobic conditions, whereas the msrA and msrB strains grew normally. Agar plate disc diffusion assays showed that all mutants displayed increased sensitivity to hydrogen peroxide, organic peroxide, superoxide, and nitrosative and disulphide stress, but quantitative cell viability assays showed that the msrA/B double mutant was markedly more sensitive to both oxidative and nitrosative stress. All of the stress-sensitivity phenotypes observed for the msrB mutant were restored to wild-type in the msrB/msrB(+) merodiploid. It is concluded that MsrA and MsrB make a significant contribution to the protection of C. jejuni against oxidative and nitrosative stress.

The wonders of flap endonucleases: structure, function, mechanism and regulation

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5-flaps. These 5-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5-flaps with exquisite specificity. FENs are paradigms for the 5 nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5 nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication.

Brønsted analysis and rate-limiting steps for the T5 flap endonuclease catalyzed hydrolysis of exonucleolytic substrates.

During replication and repair flap endonucleases (FENs) catalyze endonucleolytic and exonucleolytic (EXO) DNA hydrolyses. Altering the leaving group pK(a), by replacing the departing nucleoside with analogues, had minimal effect on k(cat)/K(M) in a T5FEN-catalyzed EXO reaction, producing a very low Brønsted coefficient, β(lg). Investigation of the viscosity dependence of k(cat)/K(M) revealed that reactions of EXO substrates are rate limited by diffusional encounter of enzyme and substrate, explaining the small β(lg). However, the maximal single turnover rate of the FEN EXO reaction also yields a near zero β(lg). A low β(lg) was also observed when evaluating k(cat)/K(M) for D201I/D204S FEN-catalyzed reactions, even though these reactions were not affected by added viscogen. But an active site K83A mutant produced a β(lg) = -1.2 ± 0.10, closer to the value observed for solution hydrolysis of phosphate diesters. The pH-maximal rate profiles of the WT and K83A FEN reactions both reach a maximum at high pH and do not support an explanation of the data that involves catalysis of leaving group departure by Lys 83 functioning as a general acid. Instead, a rate-limiting physical step, such as substrate unpairing or helical arch ordering, that occurs after substrate association must kinetically hide an inherent large β(lg). It is suggested that K83 acts as an electrostatic catalyst that stabilizes the transition state for phosphate diester hydrolysis. When K83 is removed from the active site, chemistry becomes rate limiting and the leaving group sensitivity of the FEN-catalyzed reaction is revealed.

Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions.

Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a “two-metal ion mechanism” where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg2+ ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. To investigate the positions of the ions required for chemistry, a mutant T5FEN was studied where metal 2 (M2) ligands are altered to eliminate this binding site. In contrast to WT T5FEN, the overall reaction catalyzed by D201I/D204S required two ions, but over the concentration range of Mg2+ tested, maximal rate data were fitted to a single binding isotherm. Calcium ions do not support FEN catalysis and inhibit the reactions supported by viable metal cofactors. To establish participation of ions in stabilization of enzyme-substrate complexes, dissociation constants of WT and D201I/D204S-substrate complexes were studied as a function of [Ca2+]. At pH 9.3 (maximal rate conditions), Ca2+ substantially stabilized both complexes. Inhibition of viable cofactor supported reactions of WT, and D201I/D204S T5FENs was biphasic with respect to Ca2+ and ultimately dependent on 1/[Ca2+]2. By varying the concentration of viable metal cofactor, Ca2+ ions were shown to inhibit competitively displacing two catalytic ions. Combined analyses imply that M2 is not involved in chemical catalysis but plays a role in substrate binding, and thus a two-metal ion mechanism is plausible.