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Acinetobacter baumannii is an important nosocomial bacterial pathogen. Multidrug-resistant isolates of A. baumannii are reported worldwide. Some A. baumannii isolates display resistance to nearly all antibiotics, making treatment of infections very challenging. As the need for new and effective antibiotics against A. baumannii becomes increasingly urgent, there is a need to understand the mechanisms of antibiotic resistance and virulence in this organism. In this work, comparative genomics was used to understand the mechanisms of antibiotic resistance and virulence in AB030, an extremely drug-resistant and hypervirulent strain of A. baumannii that is a representative of a recently emerged lineage of A. baumannii International Clone V. In order to characterize AB030, we carried out a genomic and phenotypic comparison with LAC-4, a previously described hyper-resistant and hypervirulent isolate. AB030 contains a number of antibiotic resistance- and virulence-associated genes that are not present in LAC-4. A number of these genes are present on mobile elements. This work shows the importance of characterizing the members of new lineages of A. baumannii in order to determine the development of antibiotic resistance and virulence in this organism.

Tuberculosis remains one of the top causes of death worldwide, and combating its spread has been severely complicated by the emergence of drug-resistance mutations, highlighting the need for more effective drugs. Despite the resistance to isoniazid (INH) arising from mutations in the katG gene encoding the catalase-peroxidase KatG, most notably the S315T mutation, this compound is still one of the most powerful first-line antitubercular drugs, suggesting further pursuit of the development of tailored INH derivatives. The N′-acylated INH derivative with a long alkyl chain (INH-C10) has been shown to be more effective than INH against the S315T variant of Mycobacterium tuberculosis, but the molecular details of this activity enhancement are still unknown. In this work, we show that INH N′-acylation significantly reduces the rate of production of both isonicotinoyl radical and isonicotinyl–NAD by wild type KatG, but not by the S315T variant of KatG mirroring the in vivo effectiveness of the compound. Restrai...

Pseudomonas brassicacearum DF41 is a biocontrol agent that suppresses disease caused by the fungal pathogen Sclerotinia sclerotiorum . A number of exometabolites are produced by DF41, including the lipopeptide sclerosin, hydrogen cyanide (HCN), and degradative enzymes. The production of these compounds is controlled at both the transcriptional and posttranscriptional levels by quorum sensing (QS) and the Gac two-component regulatory system. In order to be successful, a biocontrol agent must persist in the environment at levels sufficient for pathogen control. Bacterivorous predators, including nematodes, represent a challenge to the establishment of introduced microorganisms. In the current study, DF41 was investigated for its ability to resist predation by Caenorhabditis elegans . We discovered that this bacterium is capable of killing C. elegans through two different mechanisms: the first involves exposure to toxic metabolites, and the second entails biofilm formation on the nematode head blocking the buccal cavity. Biofilm formation on nematodes, which has been reported only for Yersinia spp. and Xenorhabdus nematophila , is dependent upon the Gac system. Biofilms were not observed when bacteria were grown on NaCl-containing medium or on C. elegans biofilm-resistant mutants. Coculturing with nematodes led to the increased expression of the pdfRI-rfiA QS genes and hcnA , which is under QS control. HCN was the most nematicidal of the exometabolites, suggesting that this bacterium can respond to predator cues and upregulate expression of toxins accordingly. In summary, DF41 is able to respond to the presence of C. elegans , and through two distinct mechanisms, it can escape predation. IMPORTANCE Pseudomonas brassicacearum DF41 can suppress fungal pathogens through a process known as biocontrol. To be successful, a biocontrol agent must be able to persist in the environment at levels sufficient for pathogen control. Predators, including the nematode Caenorhabditis elegans , represent a threat to persistence. The aim of the current study was to investigate the DF41- C. elegans interaction. We discovered that DF41 is able to escape predation through two distinct mechanisms. The first involves exposure to toxic bacterial metabolites, and the second entails the formation of a sticky coating on the nematode head, called a biofilm, which blocks feeding and causes starvation. We report here a pseudomonad forming biofilms on the C. elegans surface. When grown with C. elegans , DF41 exhibits altered gene expression and metabolite production, indicating that this bacterium can sense the presence of these predators and adjust its physiology accordingly.

Acinetobacter baumannii is an important nosocomial bacterial pathogen. Multidrug-resistant isolates of A. baumannii are reported worldwide. Some A. baumannii isolates display resistance to nearly all antibiotics, making treatment of infections very challenging. As the need for new and effective antibiotics against A. baumannii becomes increasingly urgent, there is a need to understand the mechanisms of antibiotic resistance and virulence in this organism. In this work, comparative genomics was used to understand the mechanisms of antibiotic resistance and virulence in AB030, an extremely drug-resistant and hypervirulent strain of A. baumannii that is a representative of a recently emerged lineage of A. baumannii International Clone V. In order to characterize AB030, we carried out a genomic and phenotypic comparison with LAC-4, a previously described hyper-resistant and hypervirulent isolate. AB030 contains a number of antibiotic resistance- and virulence-associated genes that are not present in LAC-4. A number of these genes are present on mobile elements. This work shows the importance of characterizing the members of new lineages of A. baumannii in order to determine the development of antibiotic resistance and virulence in this organism.

Background Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum. Methods In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations. Results In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation. Conclusions The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes., Resistance to antibiotics has become a serious problem for society, and there are increasing efforts to understand the reasons for and sources of resistance. Bacterial-encoded enzymes and transport systems, both innate and acquired, are the most frequent culprits for the development of resistance, although in Mycobacterium tuberculosis, the catalase-peroxidase, KatG, has been linked to the activation of the antitubercular drug isoniazid. While investigating a possible link between aminoglycoside antibiotics and the induction of oxidative bursts, we observed that KatG reduces susceptibility to aminoglycosides. Investigation revealed that kanamycin served as an electron donor for the peroxidase reaction, reducing the oxidized ferryl intermediates of KatG to the resting state. Loss of electrons from kanamycin was accompanied by the addition of a single oxygen atom to the aminoglycoside. The oxidized form of kanamycin proved to be less effective as an antibiotic. Kanamycin inhibited the crystallization of KatG, but the smaller, structurally related glycoside maltose did cocrystallize with KatG, providing a suggestion as to the possible binding site of kanamycin, This work was supported by Discovery Grants 2012-9600 (to P.C.L.) and 2015-05550 (to A.K.) from the Natural Sciences and Engineering Research Council (NSERC) of Canada, and by the Canada Research Chair Program (to P.C.L.). The Canadian Light Source is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. P.M.D.S. is supported by the University of Manitoba Graduate Fellowship.

In order to determine if triclosan can select for mutants of Acinetobacter baumannii ATCC 17978 that display reduced susceptibilities to antibiotics, we isolated a triclosan-resistant mutant, A. baumannii AB042, by serial passaging of A. baumannii ATCC 17978 in growth medium supplemented with triclosan. The antimicrobial susceptibility of AB042 was analyzed by the 2-fold serial dilution method. Expression of five different resistance-nodulation-division (RND) pump-encoding genes ( adeB , adeG , adeJ , A1S _ 2818 , and A1S _ 3217 ), two outer membrane porin-encoding genes ( carO and oprD ), and the MATE family pump-encoding gene abeM was analyzed using quantitative reverse transcriptase (qRT) PCR. A. baumannii AB042 exhibited elevated resistance to multiple antibiotics, including piperacillin-tazobactam, doxycycline, moxifloxacin, ceftriaxone, cefepime, meropenem, doripenem, ertapenem, ciprofloxacin, aztreonam, tigecycline, and trimethoprim-sulfamethoxazole, in addition to triclosan. Genome sequencing of A. baumannii AB042 revealed a 116 G→V mutation in fabI , the gene encoding the target enzyme for triclosan. Expression analysis of efflux pumps showed overexpression of the AdeIJK pump, and sequencing of adeN , the gene that encodes the repressor of the adeIJK operon, revealed a 73-bp deletion which would cause a premature termination of translation, resulting in an inactive truncated AdeN protein. This work shows that triclosan can select for mutants of A. baumannii that display reduced susceptibilities to multiple antibiotics from chemically distinct classes in addition to triclosan resistance. This multidrug resistance can be explained by the overexpression of the AdeIJK efflux pump.

The unusual Met-Tyr-Trp adduct composed of cross-linked side chains along with an associated mobile Arg is essential for catalase activity in catalase-peroxidases. In addition, acidic residues in the entrance channel, in particular an Asp and a Glu ∼7 and ∼15 Å, respectively, from the heme, significantly enhance catalase activity. The mechanism by which these channel carboxylates influence catalase activity is the focus of this work. Seventeen new variants with fewer and additional acidic residues have been constructed and characterized structurally and for enzymatic activity, revealing that their effect on activity is roughly inversely proportional to their distance from the heme and adduct, suggesting that the electrostatic potential of the heme cavity may be affected. A discrete group of protonable residues are contained within a 15 Å sphere surrounding the heme iron, and a computational analysis reveals that the pKa of the distal His112, alone, is modulated within the pH range of catalase activity by the remote acidic residues in a pattern consistent with its protonated form having a key role in the catalase reaction cycle. The electrostatic potential also impacts the catalatic reaction through its influence on the charged status of the Met-Tyr-Trp adduct., This work was supported by a Discovery Grant 9600 from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to P.C.L.) and the Canada Research Chair Program (to P.C.L.). The Canadian Light Source is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. We also acknowledge financial support from the Portuguese Fundaca̧ o para a Ciencia e a Tecnologia through ̂ Project UID/MULTI/00612/2013.

International audience; The location of the Trp radical and the catalytic function of the [Fe(IV)═O Trp191•+] intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr71• and Tyr236• is independent of the [Fe(IV)═O Trp191•+] intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Å from the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632–1639]. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr71• as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking [Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248–255] and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole [Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665–11674]. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction [Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557–8563]. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.

Catalase-peroxidases or KatGs can utilize organic peroxyacids and peroxides instead of hydrogen peroxide to generate the high-valent ferryl-oxo intermediates involved in the catalase and peroxidase reactions. In the absence of peroxidatic one-electron donors, the ferryl intermediates generated with a low excess (10-fold) of peroxyacetic acid (PAA) slowly decay to the ferric resting state after several minutes, a reaction that is demonstrated in this work by both stopped-flow UV-vis absorption measurements and EPR spectroscopic characterization of Burkholderia pseudomallei KatG (BpKatG). EPR spectroscopy showed that the [FeIV=O Trp330 •+], [FeIV=O Trp139 •], and [FeIV=O Trp153 •] intermediates of the peroxidase-like cycle of BpKatG (Colin, J.Wiseman, B.Switala, J.Loewen, P. C.Ivancich, A. (2009) J. Am. Chem. Soc. 131, 8557-8563), formed with a low excess of PAA at low temperature, are also generated with a high excess (1000-fold) of PAA at room temperature. However, under high excess conditions, there is a rapid conversion to a persistent [FeIV=O] intermediate. Analysis of tryptic peptides of BpKatG by mass spectrometry before and after treatment with PAA showed that specific tryptophan (including W330, W139, and W 153), methionine (including Met264 of the M-Y-W adduct), and cysteine residues are either modified with one, two, or three oxygen atoms or could not be identified in the spectrum because of other undetermined modifications. It was concluded that these oxidized residues were the source of electrons used to reduce the excess of PAA to acetic acid and return the enzyme to the ferric state. Treatment of BpKatG with PAA also caused a loss of catalase activity towards certain substrates, consistent with oxidative disruption of the M-Y-W adduct, and a loss of peroxidase activity, consistent with accumulation of the [FeIV=O] intermediate and the oxidative modification of the W330, W139, and W153. PAA, but not H2O2 or tert-butyl hydroperoxide, also caused subunit cross-linking. © 2013 American Chemical Society., This work was supported by a Discovery Grant 9600 from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to P.C.L.), the Canada Research Chair Program (to P.C.L.), the French CNRS and CEA-Saclay (UMR 8221) (to A.I.), and the CNRS Program PICS-Canada #05865 (to A.I.).

Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human per-oxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Inter-subunit disulfide bridges, Cys-206-Cys-206 and Cys-243-Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Discovery Grant 9600 from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chair Program (to P. C. L.) and by Grant BFU2012-36827 from the Ministerio de Ciencia e Innovación (MICINN) and Grant SGR2009-00327 from the Generalitat de Catalunya (to I. F.)

We report here a genome sequence for Rhodococcus sp. isolate UM008 isolated from the renal/interrenal tissue of the winter skate Leucoraja ocellata . Genome sequence analysis suggests that Rhodococcus bacteria may act in a novel mutualistic relationship with their elasmobranch host, serving as biocatalysts in the steroidogenic pathway of 1α-hydroxycorticosterone.

The Helicobacter pylori energy sensor TlpD determines tactic behaviour under low energy conditions and is important in vivo. We explored protein-protein interactions of TlpD and their impact on TlpD localisation and function. Pull-down of tagged TlpD identified protein interaction partners of TlpD, which included the chemotaxis histidine kinase CheAY2, the central metabolic enzyme aconitase (AcnB) and the detoxifying enzyme catalase (KatA). We confirmed that KatA and AcnB physically interact with TlpD. While the TlpD-dependent behavioural response appeared not influenced in the interactor mutants katA and acnB in steady-state behavioural assays, acetone carboxylase subunit (acxC) mutant behaviour was altered. TlpD was localised in a bipolar subcellular pattern in media of high energy. We observed a significant change in TlpD localisation towards the cell body in cheAY2-, catalase- or aconitase-deficient bacteria or in bacteria incubated under low energy conditions, including oxidative stress or respiratory inhibition. Inactivation of tlpD resulted in an increased sensitivity to iron limitation and oxidative stress and influenced the H. pylori transcriptome. Oxidative stress, iron limitation and overexpressing the iron-sulfur repair system nifSU altered TlpD-dependent behaviour. We propose that TlpD localisation is instructed by metabolic activity and protein interactions, and its sensory activity is linked to iron-sulfur cluster integrity.

Aims Catalase catalyzes the degradation of H2O2. Acinetobacter species have four predicted catalase genes, katA, katE, katG, and katX. The aims of the present study seek to determine which catalase(s) plays a predominant role in determining the resistance to H2O2, and to assess the role of catalase in Acinetobacter virulence. Main methods Mutants of Acinetobacter baumannii and Acinetobacter nosocomialis with deficiencies in katA, katE, katG, and katX were tested for sensitivity to H2O2, either by halo assays or by liquid culture assays. Respiratory burst of neutrophils, in response to A. nosocomialis, was assessed by chemiluminescence to examine the effects of catalase on the production of reactive oxygen species (ROS) in neutrophils. Bacterial virulence was assessed using a Galleria mellonella larva infection model. Key findings The capacities of A. baumannii and A. nosocomialis to degrade H2O2 are largely dependent on katE. The resistance of both A. baumannii and A. nosocomialis to H2O2 is primarily determined by the katG gene, although katE also plays a minor role in H2O2 resistance. Bacteria lacking both the katG and katE genes exhibit the highest sensitivity to H2O2. While A. nosocomialis bacteria with katE and/or katG were able to decrease ROS production by neutrophils, these cells also induced a more robust respiratory burst in neutrophils than did cells deficient in both katE and katG. We also found that A. nosocomialis deficient in both katE and katG was more virulent than the wildtype A. nosocomialis strain. Significance Our findings suggest that inhibition of Acinetobacter catalase may help to overcome the resistance of Acinetobacter species to microbicidal H2O2 and facilitate bacterial disinfection.

The interconversion of glycerol 3-phosphate and dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenases provides a link between carbohydrate and lipid metabolism and provides Saccharomyces cerevisiae with protection against osmotic and anoxic stress. The first structure of a glycerol-3-phosphate dehydrogenase from S. cerevisiae, GPD1, is reported at 2.45 Ã… resolution. The asymmetric unit contains two monomers, each of which is organized with N- and C-terminal domains. The N-terminal domain contains a classic Rossmann fold with the (Î’ - Î’ - Î’)2 motif typical of many NAD +-dependent enzymes, while the C-terminal domain is mainly -helical. Structural and phylogenetic comparisons reveal four main structure types among the five families of glycerol-3-phosphate and glycerol-1-phosphate dehydrogenases and reveal that the Clostridium acetobutylican protein with PDB code 3ce9 is a glycerol-1 - phosphate dehydrogenase. © 2012 International Union of Crystallography All rights reserved., This work was supported by a Discovery Grant 9600 from the Natural Sciences and Engineering Research Council of Canada, by the Canada Research Chair Program (to PCL) and by grant BFU2009-09268 from the Ministerio de Educación y Ciencia (MEC), Spain (to IF).

The main channel for H 2O 2 access to the heme cavity in large subunit catalases is twice as long as in small subunit catalases and is divided into two distinct parts. Like small subunit catalases, the 15 of the channel adjacent to the heme has a predominantly hydrophobic surface with only weak water occupancy, but the next 15 extending to the protein surface is hydrophilic and contains a complex water matrix in multiple passages. At the approximate junction of these two sections are a conserved serine and glutamate that are hydrogen bonded and associated with H 2O 2 in inactive variants. Mutation of these residues changed the dimensions of the channel, both enlarging and constricting it, and also changed the solvent occupancy in the hydrophobic, inner section of the main channel. Despite these structural changes and the prominent location of the residues in the channel, the variants exhibited less than a 2-fold change in the k cat and apparent K M kinetic constants. These results reflect the importance of the complex multi-passage structure of the main channel. Surprisingly, mutation of either the serine or glutamate to an aliphatic side chain interfered with heme oxidation to heme d. © 2012 Elsevier Inc. All rights reserved., This work was supported by a Discovery Grant 9600 from the Natural Sciences and Engineering Research Council of Canada and by the Canada Research Chair Program (to P.C.L.).

Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 A resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal +Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.

The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.

Catalase-peroxidases or KatGs from seven different organisms, including Archaeoglobus fulgidus, Bacillus stearothermophilus , Burkholderia pseudomallei , Escherichia coli , Mycobacterium tuberculosis , Rhodobacter capsulatus and Synechocystis PCC 6803, have been characterized to provide a comparative picture of their respective properties. Collectively, the enzymes exhibit similar turnover rates with the catalase and peroxidase reactions varying between 4900 and 15,900 s −1 and 8–25 s −1 , respectively. The seven enzymes also exhibited similar pH optima for the peroxidase (4.25–5.0) and catalase reactions (5.75), and high sensitivity to azide and cyanide with IC 50 values of 0.2–20 μM and 50–170 μM, respectively. The K M s of the enzymes for H 2 O 2 in the catalase reaction were relatively invariant between 3 and 5 mM at pH 7.0, but increased to values ranging from 20 to 225 mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H 2 O 2 binding to Cpd I. The catalatic k cat was 2- to 3-fold higher at pH 5 compared to pH 7, consistent with the uptake of a proton being involved in the reduction of Cpd I. The turnover rates for the INH lyase and isonicotinoyl-NAD synthase reactions, responsible for the activation of isoniazid as an anti-tubercular drug, were also similar across the seven enzymes, but considerably slower, at 0.5 and 0.002 s −1 , respectively. Only the NADH oxidase reaction varied more widely between 10 −4 and 10 −2 s −1 with the fastest rate being exhibited by the enzyme from B. pseudomallei .

Catalase-peroxidases (KatGs) are bifunctional heme proteins, belonging to the family of class I peroxidases, that are able to catalyze both catalatic and peroxidatic reactions within a peroxidase-like structure. We investigated the electronic structure of reaction intermediates of the catalytic cycle of KatGs by means of density functional theory (DFT) QM/MM calculations. The outcome was that the ionization state of the KatG-specific covalent adduct (Met264-Tyr238-Trp111) affects the radical character of compound I (Cpd I). Specifically, in the optimized structures, substantial radical character is observed on the proximal Trp330 when Tyr238 is protonated, whereas when Tyr238 is deprotonated the radical localizes on the Met+-Tyr(O -)-Trp adduct. These findings are not affected by protein thermal fluctuations, although details of the spin density distribution are affected by the geometry of the active site. Calculations provide structures in good agreement with the crystal structure of BpKatG Cpd I. They also provide an explanation for the experimental findings of the mobile and catalatic-specific residue Arg426 being 100% in conformation R in the X-ray structure of BpKatG treated with organic peroxides. The role of different Cpd I forms in the catalase and peroxidase reaction pathways is discussed. © 2007 American Chemical Society., This work was supported by Grants 2005SGR-00036 from the Generalitat de Catalunya (GENCAT), FIS2005-00655, BFU2004-06377-C02-02 from the Ministerio de Educación y Cencia (MEC), Spain, by Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P.C.L.), and by the Canadian Research Chair Program (to P.C.L). Financial support from the Juan de la Cierva program of MEC (to P.V.), the I3P program of the Spanish Research Council (to X.C.), and the F.I. fellowship program of the GENCAT (to M.A.-P.) was provided

Abscisic acid ((+)-ABA) is a phytohormone involved in the modulation of developmental processes and stress responses in plants. A chemical proteomics approach using an ABA mimetic probe was combined with in vitro assays, isothermal titration calorimetry (ITC), x-ray crystallography and in silico modelling to identify putative (+)-ABA binding-proteins in crude extracts of Arabidopsis thaliana. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was identified as a putative ABA-binding protein. Radiolabelled-binding assays yielded a Kd of 47 nM for (+)-ABA binding to spinach Rubisco, which was validated by ITC, and found to be similar to reported and experimentally derived values for the native ribulose-1,5-bisphosphate (RuBP) substrate. Functionally, (+)-ABA caused only weak inhibition of Rubisco catalytic activity (Ki of 2.1 mM), but more potent inhibition of Rubisco activation (Ki of ~ 130 μM). Comparative structural analysis of Rubisco in the presence of (+)-ABA with RuBP in the active site revealed only a putative low occupancy (+)-ABA binding site on the surface of the large subunit at a location distal from the active site. However, subtle distortions in electron density in the binding pocket and in silico docking support the possibility of a higher affinity (+)-ABA binding site in the RuBP binding pocket. Overall we conclude that (+)-ABA interacts with Rubisco. While the low occupancy (+)-ABA binding site and weak non-competitive inhibition of catalysis may not be relevant, the high affinity site may allow ABA to act as a negative effector of Rubisco activation.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Crystals of Burkholderia pseudomallei KatG retain their ability to diffract X-rays at high resolution after adjustment of the pH from 5.6 to 4.5, 6.5, 7.5, and 8.5, providing a unique view of the effect of pH on protein structure. One significant pH-sensitive change lies in the appearance of a perhydroxy group attached to the indole nitrogen of the active site Trp111 above pH 7, similar to a modification originally observed in the Ser324Thr variant of the enzyme at pH 5.6. The modification forms rapidly from molecular oxygen in the buffer with 100% occupancy after one minute of soaking of the crystal at room temperature and pH 8.5. The low temperature (4 K) ferric EPR spectra of the resting enzyme, being very sensitive to changes in the heme iron microenvironment, confirm the presence of the modification above pH 7 in native enzyme and variants lacking Arg426 or Met264 and its absence in variants lacking Trp111 or Tyr238. The indole-perhydroxy group is very likely the reactive intermediate of molecular oxygen in the NADH oxidase reaction, and Arg426 is required for its reduction. The second significant pH-sensitive change involves the buried side chain of Arg426 that changes from one predominant conformation at low pH to a second at high pH. The pH profiles of the peroxidase, catalase, and NADH oxidase reactions can be correlated with the distribution of Arg426 conformations. Other pH-induced structural changes include a number of surface-situated side chains, but there is only one change involving a displacement of main chain atoms triggered by the protonation of His53 in a deep pocket in the vicinity of the molecular 2-fold axis. © 2006 American Chemical Society., This work was supported by a grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P.C.L.), by the Canadian Research Chair Program (to P.C.L.) and by fellowship EX-2003-0866 from the Ministerio de Educación Cultura y Deporte, Spain (to X.C.)

The catalase reaction of catalase-peroxidases involves catalase-specific features built into a peroxidase core. An arginine, 20 Å from the active-site heme, acts as a molecular switch moving between two conformations, one that activates heme oxidation and one that activates oxoferryl heme reduction by H2O2, facilitating the catalatic pathway in a peroxidase. The influence of the arginine is imparted to the heme through its association with or dissociation from a tyrosinate that modulates reactivity through a Met-Tyr-Trp crosslinked adduct and a π electron interaction of the heme with the adduct Trp. © 2005 European Molecular Biology Organization., This work was supported by grant BIO2002-04419 from Ministerio de Ciencia y Technologia, Spain (to I.F.), by grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P.C.L.), by the Canada Research Chair Program (to P.C.L.) and by fellowship EX-2003-0866 from the Ministerio de Educación Cultura y Deporte, Spain (to X.C.)

Vc-NhaD is a Na(+)/H(+) antiporter from Vibrio cholerae with a sharp maximum of activity at pH approximately 8.0. NhaD homologues are present in many bacteria as well as in higher plants. However, very little is known about structure-function relations in NhaD-type antiporters. In this work 14 conserved polar residues associated with putative transmembrane segments of Vc-NhaD have been screened for their possible role in the ion translocation and pH regulation of Vc-NhaD. Substitutions S150A, D154G, N155A, N189A, D199A, T201A, T202A, S389A, N394G, S428A, and S431A completely abolished the Vc-NhaD-mediated Na(+)-dependent H(+) transfer in inside-out membrane vesicles. Substitutions T157A and S428A caused a significant increase of apparent K(m) values for alkali cations, with the K(m) for Li(+) elevated more than that for Na(+), indicating that Thr-157 and Ser-428 are involved in alkali cation binding/translocation. Of six conserved His residues, mutation of only His-93 and His-210 affected the Na(+)(Li(+))/H(+) antiport, resulting in an acidic shift of its pH profile, whereas H93A also caused a 7-fold increase of apparent K(m) for Na(+) without affecting the K(m) for Li(+). These data suggest that side chains of His-93 and His-210 are involved in proton binding and that His-93 also contributes to the binding of Na ions during the catalytic cycle. These 15 residues are clustered in three distinct groups, two located at opposite sides of the membrane, presumably facilitating the access of substrate ions to the third group, a putative catalytic site in the middle of lipid bilayer. The distribution of these key residues in Vc-NhaD molecule also suggests that transmembrane segments IV, V, VI, X, XI, and XII are situated close to one another, creating a transmembrane relay of charged/polar residues involved in the attraction, coordination, and translocation of transported cations.

More than 300 catalase sequences are now available, divided among monofunctional catalases (> 225), bifunctional catalase-peroxidases (> 50) and manganese-containing catalases (> 25). When combined with the recent appearance of crystal structures from at least two representatives from each of these groups (nine from the monofunctional catalases), valuable insights into the catalatic reaction mechanism in its various forms and into catalase evolution have been gained. The structures have revealed an unusually large number of modifications unique to catalases, a result of interacting with reactive oxygen species. Biochemical and physiological characterization of catalases from many different organisms has revealed a surprisingly wide range of catalatic efficiencies, despite similar sequences. Catalase gene expression in micro-organisms generally is controlled either by sensors of reactive oxygen species or by growth phase regulons, although the detailed mechanisms vary considerably., Preparation of this manuscript was supported by Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada to P. C. Loewen

The electron density maps of the catalase-peroxidase from Burkholderia pseudomallei (BpKatG) presented two unusual covalent modifications. A covalent structure linked the active site Trp111 with Tyr238 and Tyr238 with Met264, and the heme was modified, likely by a perhydroxy group added to the vinyl group on ring I. Mass spectrometry analysis of tryptic digests of BpKatG revealed a cluster of ions at m/z 6585, consistent with the fusion of three peptides through Trp111, Tyr 238, and Met264, and a cluster at m/z ∼4525, consistent with the fusion of two peptides linked through Trp111 and Tyr238. MS/MS analysis of the major ions at m/z 4524 and 4540 confirmed the expected sequence and suggested that the multiple ions in the cluster were the result of multiple oxidation events and transfer of CH 3-S to the tyrosine. Neither cluster of ions at m/z 4525 or 6585 was present in the spectrum of a tryptic digest of the W111F variant of BpKatG. The spectrum of the tryptic digest of native BpKatG also contained a major ion for a peptide in which Met264 had been converted to homoserine, consistent with the covalent bond between Tyr238 and Met 264 being susceptible to hydrolysis, including the loss of the CH3-S from the methionine. Analysis of the tryptic digests of hydroperoxidase I (KatG) from Escherichia coli provided direct evidence for the covalent linkage between Trp105 and Tyr226 and indirect evidence for a covalent linkage between Tyr226 and Met 252. Tryptic peptide analysis and N-terminal sequencing revealed that the N-terminal residue of BpKatG is Ser22., This work was supported by Grants BIO099-0865 and BIO2002-04419 from Direccion General de Investigacion Ciencia y Technologia (to I. F.) and Grant OGP9600 from the Natural Sciences and Engineering Research Council of Canada (to P. C. L.)

The past decade has produced an increasing number of reports on horizontal gene transfer between prokaryotic organisms. Only recently, with the flood of available whole genome sequence data and a renewed intensity of the debate about the universal tree of life, a very few reports on lateral gene transfer (LGT) from prokaryotes into the Eukaryota have been published. We have investigated and report here on the molecular evolution of the gene families that encode catalatic hydroperoxidases. We have found that this process included not only frequent horizontal gene transfer among prokaryotes but also several lateral gene transfer events between bacteria and fungi and between bacteria and the protistan ancestor of the alga/plant lineage.

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases.

The Vc-NhaD is an Na + /H + antiporter from Vibrio cholerae belonging to a new family of bacterial Na + /H + antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu 100 Ala, Glu 251 Ala, Glu 342 Ala, and Asp 393 Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp 344 Ala, Asp 344 Asn, and Thr 345 Ala caused a complete loss of both Na + /H + and Li + /H + antiport activity whereas the Asp 344 Glu variant exhibited reduced Na + /H + and Li + /H + antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na + /H + antiport. We discuss the possible role of Asp 344 and Thr 345 in the functioning of Vc-NhaD. D 2002 Elsevier Science B.V. All rights reserved.

A mer-lux bioreporter was used to study uptake of inorganic mercury, Hg(II), at trace concentrations by two facultatively anaerobic bacterial species, Vibrio anguillarumand Escherichia coli. Uptake of Hg(II) by these bacteria appeared to be facilitated, rather than by passive diffusion. Three lines of evidence support this conclusion. First, under anaerobic conditions Hg(II) uptake was greatly decreased compared with aerobic conditions, even though the chemical composition of the medium was identical except for the lack of oxygen (i.e., no reducing agents were used). Second, the uptake of Hg(II) under anaerobic conditions was not proportional to the abundance of lipophilic Hg species but was dependent on the total concentration of Hg in the samples. Third, at trace Hg(II) concentrations and under anaerobic conditions, Hg(II) uptake was enhanced by the addition of yeast extract and a variety of low molecular weight organic acids. In addition to demonstrating that Hg(II) uptake by these bacteria had the characteristics of facilitated transport, these lines of evidence also support the conclusion that processes under regulatory control of the cell affected Hg(II) uptake. If these findings apply to other bacteria as well, they mean that current models of Hg(II) uptake by microorganisms in aquatic systems, which are based solely on the role of lipophilic Hg species and passive diffusion, will need to be reconsidered.

We report the 4.3-Mbp genome sequence of a blood isolate of Acinetobacter baumannii strain AB030.

We report here the 3.8-Mbp genome sequence of a blood isolate of Acinetobacter baumannii strain AB031.

Pseudomonas chlororaphis strain PA23 is a plant-beneficial bacterium that is able to suppress disease caused by the fungal pathogen Sclerotinia sclerotiorum through a process known as biological control. Here we present a 7.1-Mb assembly of the PA23 genome.

Pseudomonas brassicacearum DF41, a Gram-negative soil bacterium, is able to suppress the fungal pathogen Sclerotinia sclerotiorum through a process known as biological control. Here, we present a 6.8-Mb assembly of its genome, which is the second fully assembled genome of a P. brassicacearum strain.

Bacillus pumilus is a Gram-positive, rod-shaped, aerobic bacterium isolated from the soil. B. pumilus strain B6033 was originally selected as a biocatalyst for the stereospecific oxidation of β-lactams. Here, we present a 3.8-Mb assembly of its genome, which is the second fully assembled genome of a B. pumilus strain.

Isonicotinic acid hydrazide (isoniazid or INH) is a front line antitubercular pro-drug that is converted to its active form, isonicotinyl-NAD, by the bacterial catalase-peroxidase KatG. Understanding the role of KatG in the INH activation process has been hampered by a lack of knowledge of the actual drug binding site. In this work, we have investigated the binding of INH in the main access channel of KatG with a combination of molecular dynamics, using an enhanced-sampling technique (metadynamics), X-ray crystallography, and site-directed mutagenesis. The metadynamics simulations show that there are several weak drug binding sites along the access channel. Moreover, the simulations evidence that complete entrance to the heme active site is impeded by an aspartate residue (D141) located above the heme. This has been confirmed by structural and functional analysis of the D141A mutant, leading to the first X-ray crystallography evidence of INH at the heme access channel. © 2014 American Chemical Society., Financial support was provided by MINECO (Grants CTQ2011-25871 and BFU2012-36827), GENCAT (Grant 2009SGR-1309), NSERC (Discovery Grant 9600), and the Canada Research Chair Program (to P.C.L.)

9 pages, 6 figures, 3 tables.-- PMID: 10488114 [PubMed]., The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered., This work was supported by Direccion General de Investigacion Ciencia Y Technologia Grant PB95-0218 and the European Union through the Human Capital Mobility Project to Large Installations Project (contract HGE-CT93–0040) (in Barcelona), Natural Sciences and Engineering Research Council of Canada Grant RGP9600 (in Winnipeg), and NATO Collaborative Research Grant SA-5-2-05 (to P. C. L. and I. F.).

The catalase-peroxidase hydroperoxidase I of Escherichia coli has been confirmed to be located in the cytoplasm using two independent methods. Catalase activity was found predominantly (>95%) in the cytoplasmic fraction following spheroplast formation. The cytoplasmic enzyme glucose-6-phosphate dehydrogenase and the periplasmic enzyme alkaline phosphatase were used as controls. The second method of immunogold staining for the enzyme in situ revealed an even distribution of the enzyme across the cell.

Seventy-four catalase protein sequences, including 29 bacterial, 8 fungal, 7 animal, and 30 plant sequences, were compiled, and 70 were used for phylogenetic reconstruction. The core of the resulting tree revealed unique, separate groups of plant and animal catalases, two groups of fungal catalases, and three groups of bacterial catalases. The only overlap of kingdoms occurred within one branch and involved fungal and bacterial large-subunit enzymes. The other fungal branch was closely linked to the group of animal enzymes. Group I bacterial catalases were more closely related to the plant enzymes and contained such diverse taxa as the Gram-positive Listeria seeligeri, Deinocococcus radiodurans, and gamma-proteobacteria. Group III bacterial sequences were more closely related to fungal and animal sequences and included enzymes from a broad range of bacteria including high- and low-GC Gram positives, proteobacteria, and a bacteroides species. Group II was composed of large-subunit catalases from diverse sources including Gram positives (low-GC Bacilli and high-GC Mycobacteria), proteobacteria, and species of the filamentous fungus Aspergillus. These data can be interpreted in terms of two gene duplication events that produced a minimum of three catalase gene family members that subsequently evolved in response to environmental demands. Horizontal gene transfer may have been responsible for the group II mixture of bacterial and fungal large-subunit catalases.

8 pages, 7 figures, 1 table.-- PMID: 9144772 [PubMed].-- PMCID: PMC2143697., A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed., n. Thwiso rk was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC (OGP9600) to P.C.L. and a postgraduate scholarship from NSERC to A.H. Work in Barcelona was supported by grants PB92-0707 and PB95-0218 to I.F. J.B. was the recipient of a doctoral fellowship from the Generalitat de Catalunya. Data collection in Hamburg was supporbteyd the Human Capital Mobility Project on contract CHGE-CT93-0040

The first structure of a ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from a pulse crop is reported. Rubisco was purified from Pisum sativum (garden pea) and diffraction-quality crystals were obtained by hanging-drop vapour diffusion in the presence of the substrate ribulose 1,5-bisphosphate. X-ray diffraction data were recorded to 2.20 Ã… resolution from a single crystal at the Canadian Light Source. The overall quaternary structure of non-activated P. sativum Rubisco highlights the conservation of the form I Rubisco hexadecameric complex. The electron density places the substrate in the active site at the interface of the large-subunit dimers. Lys201 in the active site is not carbamylated as expected for this non-activated structure. Some heterogeneity in the small-subunit sequence is noted, as well as possible variations in the conformation and contacts of ribulose 1,5-bisphosphate in the large-subunit active sites. Overall, the active-site conformation most closely correlates with the 'closed' conformation observed in other substrate/inhibitor- bound Rubisco structures. © 2013 International Union of Crystallography. All rights reserved., This work was supported by the National Research Council of Canada (to MCL), the Canada Research Chair Program (to PCL) and the Natural Sciences and Engineering Research Council Discovery Grants Program (to both MCL and PCL). This manuscript represents NRC Communication No. 54669.

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

Escherichia coli produces two catalases or hydroperoxidases, HPI and HPII. HPI is a bifunctional catalase-peroxidase active as a tetramer of identical 80049-Da subunits encoded by katG. The expression of katG is controlled at the basal level by sigma s (KatF), and its induction by H2O2 is regulated by OxyR. HPII is a monofunctional catalase active as a tetramer of identical 84118-Da subunits encoded by katE. The induction of katE expression in the stationary phase is controlled by sigma s. The core of HPII is similar in sequence to other catalases including the conservation of several residues that have been implicated as playing a catalytic role, His128, Asn201, Ser167 and Tyr415. These residues have served as targets for site-directed mutagenesis in a study that has demonstrated their role in the catalytic mechanism of HPII. In addition, the two Cys residues in HPII have been targeted in a similar study revealing that they do not have a catalytic role, but that Cys438 is blocked by a novel modification. Despite many structural similarities to bovine liver catalase, the heme component of HPII has proved to be quite different. The presence of a cis heme d was determined spectrally and chromatographically, and the inability of certain mutants to generate the modified heme revealed that it was HPII itself that was catalysing the oxidation of heme b to heme d. The recent solution of the crystal structure of HPII and mass spectrometry have revealed that the heme d bound to HPII is a spirolactone structure with a cis orientation of the oxygens on the proximal side of the heme. This has created the problem of explaining how the oxidation of the heme can occur on the opposite side of the heme ring, remote from the catalytic residues.

Background: Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms. It serves, in part, to protect the cell from the toxic effects of small peroxides. Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties. HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, mono-functional catalase containing one cis -heme d prosthetic group per subunit of 753 residues. Results The crystal structure of catalase HPII from E. coli has been determined to 2.8 a resolution. The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry. In the model built, that includes residues 27–753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained. The crystallographic agreement R-factor is 20.1% for 58477 reflections in the resolution shell 8.0–2.8 a. Conclusion Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.

Site-directed mutagenesis of the katE gene of Escherichia coli was used to change, individually and in combination, Cys438 and Cys669 to serine in catalase HPII. The Cys438Ser mutation caused a 30% reduction in the specific activity of the enzyme, whereas the Cys669Ser mutation did not affect enzyme activity. The titration of free sulfhydryl groups in HPII revealed that Cys669 was reactive whereas Cys438 was unreactive. Properties of the modification on Cys438 included alkali lability, insensitivity to methyl-amine, hydroxylamine or reducing agents, and a mass determined by mass spectrometry to be approximately 43±2 Da. A hemithioacetal structure is consistent with these properties. Although free sulfhydryl groups do not play a significant role in the stability or catalytic mechanism of HPII, the sulfhydryl agent 2-mercaptoethanol caused a 50% inactivation of HPII along with an irreversible change in the absorption spectrum of the protein. Other sulfhydryl agents, including dithiothreitol, cysteine and glutathione, and the organic peroxide, t-butylhydroperoxide, which cannot directly access the active site, do not affect HPII activity, but they do cause a small reversible change in the absorption spectrum, possibly by a mechanism involving superoxide.

Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal (+)Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.

1. NADPH binds to bovine catalase and to yeast catalases A and T, but not to Escherichia coli catalase HPII. The association was demonstrated using chromatography and fluorimetry. Bound NADPH fluoresces in a similar way to NADPH in solution. 2. Bound NADPH protects bovine and yeast catalases against forming inactive peroxide compound II either via endogenous reductant action or by ferrocyanide reduction during catalytic activity in the presence of slowly generated peroxide. 3. Bound NADPH reduces neither compound I nor compound II of catalase. It apparently reacts with an intermediate formed during the decay of compound I to compound II; this postulated intermediate is an immediate precursor of stable compound II either when the latter is formed by endogenous reductants or when ferrocyanide is used. It represents therefore a new type of hydrogen donor that is not included in the original classification of Keilin and Nicholls [Keilin, D. and Nicholls, P. (1958) Biochim. Biophys. Acta 29, 302-307] 4. A model for NADPH action is presented in which concerted reduction of the ferryl iron and of a neighbouring protein free radical is responsible for the observed NADPH effects. The roles of migrant radical species in mammalian and yeast catalases are compared with similar events in metmyoglobin and cytochrome c peroxidase reactions with peroxides.