Global approaches for protein thiol redox state detection.

Due to its nucleophilicity, the thiol group of cysteine is chemically very versatile. Hence, cysteine often has important functions in a protein, be it as the active site or, in extracellular proteins, as part of a structural disulfide. Within the cytosol, cysteines are typically reduced. But the nucleophilicity of its thiol group makes it also particularly prone to post-translational oxidative modifications. These modifications often lead to an alteration of the function of the affected protein and are reversible in vivo, e.g. by the thioredoxin and glutaredoxin system. The in vivo-reversible nature of these modifications and their genesis in the presence of localized high oxidant levels led to the paradigm of thiol-based redox regulation, the adaptation, and modulation of the cellular metabolism in response to oxidative stimuli by thiol oxidation in regulative proteins. Consequently, the proteomic study of these oxidative posttranslational modifications of cysteine plays an indispensable role in redox biology.

The oxidative stress response, in particular the katY gene, is temperature-regulated in Yersinia pseudotuberculosis.

Pathogenic bacteria, such as Yersinia pseudotuberculosis encounter reactive oxygen species (ROS) as one of the first lines of defense in the mammalian host. In return, the bacteria react by mounting an oxidative stress response. Previous global RNA structure probing studies provided evidence for temperature-modulated RNA structures in the 5'-untranslated region (5'-UTR) of various oxidative stress response transcripts, suggesting that opening of these RNA thermometer (RNAT) structures at host-body temperature relieves translational repression. Here, we systematically analyzed the transcriptional and translational regulation of ROS defense genes by RNA-sequencing, qRT-PCR, translational reporter gene fusions, enzymatic RNA structure probing and toeprinting assays. Transcription of four ROS defense genes was upregulated at 37°C. The trxA gene is transcribed into two mRNA isoforms, of which the most abundant short one contains a functional RNAT. Biochemical assays validated temperature-responsive RNAT-like structures in the 5'-UTRs of sodB, sodC and katA. However, they barely conferred translational repression in Y. pseudotuberculosis at 25°C suggesting partially open structures available to the ribosome in the living cell. Around the translation initiation region of katY we discovered a novel, highly efficient RNAT that was primarily responsible for massive induction of KatY at 37°C. By phenotypic characterization of catalase mutants and through fluorometric real-time measurements of the redox-sensitive roGFP2-Orp1 reporter in these strains, we revealed KatA as the primary H2O2 scavenger. Consistent with the upregulation of katY, we observed an improved protection of Y. pseudotuberculosis at 37°C. Our findings suggest a multilayered regulation of the oxidative stress response in Yersinia and an important role of RNAT-controlled katY expression at host body temperature.

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding in Escherichia coli.

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo "steady state" redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was almost fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the steady state periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA.

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

The Interplay between S-Glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts.

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca2+ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

Metaproteomic Discovery and Characterization of a Novel Lipolytic Enzyme From an Indian Hot Spring.

Lipolytic enzymes are produced by animals, plants and microorganisms. With their chemo-, regio-, and enantio-specific characteristics, lipolytic enzymes are important biocatalysts useful in several industrial applications. They are widely used in the processing of fats and oils, detergents, food processing, paper and cosmetics production. In this work, we used a new functional metaproteomics approach to screen sediment samples of the Indian Bakreshwar hot spring for novel thermo- and solvent-stable lipolytic enzymes. We were able to identify an enzyme showing favorable characteristics. DS-007 showed high hydrolytic activity with substrates with shorter chain length (C10, significantly less hydrolytic activity was observed. A preference for short chain acyl groups is characteristic for esterases, suggesting that DS-007 is an esterase. Consistent with the high temperature at its site of isolation, DS-007 showed a temperature optimum at 55°C and retained 80% activity even after prolonged exposure to temperatures as high as 60°C. The enzyme showed optimum activity at pH 9.5, with more than 50% of its optimum activity between pH 8.0 and pH 9.5. DS-007 also exhibited tolerance toward organic solvents at a concentration of 1% (v/v). One percent of methanol increased the activity of DS-007 by 40% in comparison to the optimum conditions without solvent. In the presence of 10% methanol, DMSO or isopropanol DS-007 still showed around 50% activity. This data indicates that DS-007 is a temperature- and solvent-stable thermophilic enzyme with reasonable activity even at lower temperatures as well as a catalyst that can be used at a broad range of pH values with an optimum in the alkaline range, showing the adaptation to the habitat's temperature and alkaline pH.

Hypochlorous acid-modified human serum albumin suppresses MHC class II - dependent antigen presentation in pro-inflammatory macrophages.

Macrophages are innate immune cells that internalize and present exogenous antigens to T cells via MHC class II proteins. They operate at sites of infection in a highly inflammatory environment, generated in part by reactive oxygen species, in particular the strong oxidant hypochlorous acid (HOCl) produced in the neutrophil respiratory burst. HOCl effectively kills a broad range of pathogens but can also contribute to host tissue damage at sites of inflammation. To prevent tissue injury, HOCl is scavenged by human serum albumin (HSA) and other plasma proteins in interstitial fluids, leading to the formation of variously modified advanced oxidation products (AOPPs) with pro-inflammatory properties. Previously, we showed that HOCl-mediated N-chlorination converts HSA and other plasma proteins into efficient activators of the phagocyte respiratory burst, but the role of these AOPPs in antigen presentation by macrophages remained unclear. Here, we show that physiologically relevant amounts of N-chlorinated HSA can strongly impair the capacity of THP-1-derived macrophages to present antigens to antigen-specific T cells via MHC class II proteins at multiple stages. Initially, N-chlorinated HSA inhibits antigen internalization by converting antigens into scavenger receptor (SR) ligands and competing with the modified antigens for binding to SR CD36. Later steps of antigen presentation, such as intracellular antigen processing and MHC class II expression are negatively affected, as well. We propose that impaired processing of pathogens or exogenous antigens by immune cells at an initial stage of infection prevents antigen presentation in an environment potentially hostile to cells of the adaptive immune response, possibly shifting it towards locations removed from the actual insult, like the lymph nodes. On the flip side, excessive retardation or complete inhibition of antigen presentation by N-chlorinated plasma proteins could contribute to chronic infection and inflammation.

Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

Redox regulation in host-pathogen interactions: thiol switches and beyond.

Our organism is exposed to pathogens on a daily basis. Owing to this age-old interaction, both pathogen and host evolved strategies to cope with these encounters. Here, we focus on the consequences of the direct encounter of cells of the innate immune system with bacteria. First, we will discuss the bacterial strategies to counteract powerful reactive species. Our emphasis lies on the effects of hypochlorous acid (HOCl), arguably the most powerful oxidant produced inside the phagolysosome of professional phagocytes. We will highlight individual examples of proteins in gram-negative bacteria activated by HOCl via thiol-disulfide switches, methionine sulfoxidation, and N-chlorination of basic amino acid side chains. Second, we will discuss the effects of HOCl on proteins of the host. Recent studies have shown that both host and bacteria address failing protein homeostasis by activation of chaperone-like holdases through N-chlorination. After discussing the role of individual proteins in the HOCl-defense, we will turn our attention to the examination of effects on host and pathogen on a systemic level. Recent studies using genetically encoded redox probes and redox proteomics highlight differences in redox homeostasis in host and pathogen and give first hints at potential cellular HOCl signaling beyond thiol-disulfide switch mechanisms.

The effects of neutrophil-generated hypochlorous acid and other hypohalous acids on host and pathogens.

Neutrophils are predominant immune cells that protect the human body against infections by deploying sophisticated antimicrobial strategies including phagocytosis of bacteria and neutrophil extracellular trap (NET) formation. Here, we provide an overview of the mechanisms by which neutrophils kill exogenous pathogens before we focus on one particular weapon in their arsenal: the generation of the oxidizing hypohalous acids HOCl, HOBr and HOSCN during the so-called oxidative burst by the enzyme myeloperoxidase. We look at the effects of these hypohalous acids on biological systems in general and proteins in particular and turn our attention to bacterial strategies to survive HOCl stress. HOCl is a strong inducer of protein aggregation, which bacteria can counteract by chaperone-like holdases that bind unfolding proteins without the need for energy in the form of ATP. These chaperones are activated by HOCl through thiol oxidation (Hsp33) or N-chlorination of basic amino acid side-chains (RidA and CnoX) and contribute to bacterial survival during HOCl stress. However, neutrophil-generated hypohalous acids also affect the host system. Recent studies have shown that plasma proteins act not only as sinks for HOCl, but get actively transformed into modulators of the cellular immune response through N-chlorination. N-chlorinated serum albumin can prevent aggregation of proteins, stimulate immune cells, and act as a pro-survival factor for immune cells in the presence of cytotoxic antigens. Finally, we take a look at the emerging role of HOCl as a potential signaling molecule, particularly its role in neutrophil extracellular trap formation.

Comparison of Proteomic Responses as Global Approach to Antibiotic Mechanism of Action Elucidation.

New antibiotics are urgently needed to address the mounting resistance challenge. In early drug discovery, one of the bottlenecks is the elucidation of targets and mechanisms. To accelerate antibiotic research, we provide a proteomic approach for the rapid classification of compounds into those with precedented and unprecedented modes of action. We established a proteomic response library of Bacillus subtilis covering 91 antibiotics and comparator compounds, and a mathematical approach was developed to aid data analysis. Comparison of proteomic responses (CoPR) allows the rapid identification of antibiotics with dual mechanisms of action as shown for atypical tetracyclines. It also aids in generating hypotheses on mechanisms of action as presented for salvarsan (arsphenamine) and the antirheumatic agent auranofin, which is under consideration for repurposing. Proteomic profiling also provides insights into the impact of antibiotics on bacterial physiology through analysis of marker proteins indicative of the impairment of cellular processes and structures. As demonstrated for trans-translation, a promising target not yet exploited clinically, proteomic profiling supports chemical biology approaches to investigating bacterial physiology.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

Allicin, a natural antimicrobial defence substance from garlic, inhibits DNA gyrase activity in bacteria.

Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests. Allicin is a reactive sulfur species (RSS) that oxidizes accessible cysteines in glutathione and proteins. We used a differential isotopic labelling method (OxICAT) to identify allicin targets in the bacterial proteome. We compared the proteomes of allicin-susceptible Pseudomonas fluorescens Pf0-1 and allicin-tolerant PfAR-1 after a sublethal allicin exposure. Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation. Protein oxidation increased after exposure to allicin, and only 50% of proteins from allicin-susceptible Pf0-1, but 65% from allicin-tolerant PfAR-1, remained less than 20% oxidised. DNA gyrase was identified as an allicin target. Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria. After allicin treatment the degree of Cys433 oxidation increased to 55% in susceptible Pf0-1 but only to 10% in tolerant PfAR-1. Allicin inhibited E. coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid. Purified PfAR-1 DNA gyrase was inhibited to greater extent by allicin in vitro than the Pf0-1 enzyme. Substituting PfAR-1 GyrA into Pf0-1 rendered the exchange mutants more susceptible to allicin than the Pf0-1 wild type. Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-tolerant PfAR-1 background, rather than the PfAR-1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0-1 GyrA subunit. DNA gyrase is a target for medicinally important antibiotics; thus, allicin and its analogues may have potential to be developed as gyrase inhibitors, either alone or in conjunction with other therapeutics.

Activation leads to a significant shift in the intracellular redox homeostasis of neutrophil-like cells.

Neutrophils produce a cocktail of oxidative species during the so-called oxidative burst to attack phagocytized bacteria. However, little is known about the neutrophils' redox homeostasis during the oxidative burst and there is currently no consensus about the interplay between oxidative species and cellular signaling, e.g. during the initiation of the production of neutrophil extracellular traps (NETs). Using the genetically encoded redox sensor roGFP2, expressed in the cytoplasm of the neutrophil-like cell line PLB-985, we saw that stimulation by both PMA and E. coli resulted in oxidation of the thiol residues in this probe. In contrast to the redox state of phagocytized bacteria, which completely breaks down, the neutrophils' cytoplasmic redox state switched from its intital -318 ±â€¯6 mV to a new, albeit higher oxidized, steady state of -264 ±â€¯5 mV in the presence of bacteria. This highly significant oxidation of the cytosol (p value = 7 × 10-5) is dependent on NOX2 activity, but independent of the most effective thiol oxidant produced in neutrophils, MPO-derived HOCl. While the shift in the intracellular redox potential is correlated with effective NETosis, it is, by itself not sufficient: Inhibition of MPO, while not affecting the cytosolic oxidation, significantly decreased NETosis. Furthermore, inhibition of PI3K, which abrogates cytosolic oxidation, did not fully prevent NETosis induced by phagocytosis of bacteria. Thus, we conclude that NET-formation is regulated in a multifactorial way, in part by changes of the cytosolic thiol redox homeostasis in neutrophils, depending on the circumstance under which the generation of NETs was initiated.

Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote.

Understanding the in vivo redox biology of cells is a complex albeit important biological problem. Studying redox processes within living cells without physical disruption or chemical modifications is essential in determining the native redox states of cells. In this study, the previously characterized reduction-oxidation sensitive green fluorescent protein (roGFP2) was used to elucidate the redox changes of the genetically engineered Escherichia coli strain, SHuffle. SHuffle cells were demonstrated to be under constitutive oxidative stress and responding transcriptionally in an OxyR-dependent manner. Using roGFP2 fused to either glutathione (GSH)- or hydrogen peroxide (H2O2)- sensitive proteins (glutaredoxin 1 or Orp1), the cytosolic redox state of both wild type and SHuffle cells based on GSH/GSSG and H2O2 pools was measured. These probes open the path to in vivo studies of redox changes and genetic selections in prokaryotic hosts.

N-chlorination mediates protective and immunomodulatory effects of oxidized human plasma proteins.

Hypochlorous acid (HOCl), a powerful antimicrobial oxidant, is produced by neutrophils to fight infections. Here, we show that N-chlorination, induced by HOCl concentrations encountered at sites of inflammation, converts blood plasma proteins into chaperone-like holdases that protect other proteins from aggregation. This chaperone-like conversion was reversible by antioxidants and was abrogated by prior methylation of basic amino acids. Furthermore, reversible N-chlorination of basic amino acid side chains is the major factor that converts plasma proteins into efficient activators of immune cells. Finally, HOCl-modified serum albumin was found to act as a pro-survival molecule that protects neutrophils from cell death induced by highly immunogenic foreign antigens. We propose that activation and enhanced persistence of neutrophils mediated by HOCl-modified plasma proteins, resulting in the increased and prolonged generation of ROS, including HOCl, constitutes a potentially detrimental positive feedback loop that can only be attenuated through the reversible nature of the modification involved.

The molecular chaperone Hsp33 is activated by atmospheric-pressure plasma protecting proteins from aggregation.

Non-equilibrium atmospheric-pressure plasmas are an alternative means to sterilize and disinfect. Plasma-mediated protein aggregation has been identified as one of the mechanisms responsible for the antibacterial features of plasma. Heat shock protein 33 (Hsp33) is a chaperone with holdase function that is activated when oxidative stress and unfolding conditions coincide. In its active form, it binds unfolded proteins and prevents their aggregation. Here we analyse the influence of plasma on the structure and function of Hsp33 of Escherichia coli using a dielectric barrier discharge plasma. While most other proteins studied so far were rapidly inactivated by atmospheric-pressure plasma, exposure to plasma activated Hsp33. Both, oxidation of cysteine residues and partial unfolding of Hsp33 were observed after plasma treatment. Plasma-mediated activation of Hsp33 was reversible by reducing agents, indicating that cysteine residues critical for regulation of Hsp33 activity were not irreversibly oxidized. However, the reduction yielded a protein that did not regain its original fold. Nevertheless, a second round of plasma treatment resulted again in a fully active protein that was unfolded to an even higher degree. These conformational states were not previously observed after chemical activation with HOCl. Thus, although we could detect the formation of HOCl in the liquid phase during plasma treatment, we conclude that other species must be involved in plasma activation of Hsp33. E. coli cells over-expressing the Hsp33-encoding gene hslO from a plasmid showed increased survival rates when treated with plasma while an hslO deletion mutant was hypersensitive emphasizing the importance of protein aggregation as an inactivation mechanism of plasma.

Quantifying changes in the bacterial thiol redox proteome during host-pathogen interaction.

Phagocyte-derived production of a complex mixture of different oxidants is a major mechanism of the host defense against microbial intruders. On the protein level, a major target of these oxidants is the thiol group of the amino acid cysteine in proteins. Oxidation of thiol groups is a widespread regulatory post-translational protein modification. It is used by bacteria to respond to and to overcome oxidative stress. Numerous redox proteomic studies have shown that protein thiols in bacteria, such as Escherichia coli react towards a number of oxidants in specific ways. However, our knowledge about protein thiols in bacteria exposed to the complex mixture of oxidants encountered in the phagolysosome is still limited. In this study, we used a quantitative redox proteomic method (OxICAT) to assess the in vivo thiol oxidation status of phagocytized E. coli. The majority (65.5%) of identified proteins harbored thiols that were significantly oxidized (> 30%) upon phagocytosis. A substantial number of these proteins are from major metabolic pathways or are involved in cell detoxification and stress response, suggesting a systemic breakdown of the bacterial cysteine proteome in phagocytized bacteria. 16 of the oxidized proteins provide E. coli with a significant growth advantage in the presence of H2O2, when compared to deletion mutants lacking these proteins, and 11 were shown to be essential under these conditions.

Corrigendum: Characterization of ML-005, a Novel Metaproteomics-Derived Esterase.

[This corrects the article DOI: 10.3389/fmicb.2018.01925.].

Loss of a conserved salt bridge in bacterial glycosyl hydrolase BgIM-G1 improves substrate binding in temperate environments.

Salt bridges are the strongest electrostatic interactions in proteins. They substantially contribute to a protein's structural stability. Thus, mutations of salt bridges are typically selected against. Here, we report on the evolutionary loss of a highly conserved salt bridge in the GH1 family glycosyl hydrolase BglM-G1. BglM-G1's gene was found in the bacterial metagenome of a temperate, seasonally cold marine habitat. In BglM-G1, arginine 75 is replaced by a histidine. While fully retaining ß-glucosidase activity, BglM-G1 is less heat stable than an H75R variant, in which the salt bridge was artificially re-introduced. However, the K m toward its substrates was lower in wild type, leading to an overall higher catalytic efficiency. Our results indicate that this loss of the salt bridge leads to higher flexibility in BglM-G1's active site, trading structural stability at high temperatures, a trait not needed in a temperate, seasonally cold habitat, for a more effective catalytic activity.