scholarly journals Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

2004 ◽  
Vol 279 (44) ◽  
pp. 46082-46095 ◽  
Author(s):  
Christa Jakopitsch ◽  
Anabella Ivancich ◽  
Florian Schmuckenschlager ◽  
Anuruddhika Wanasinghe ◽  
Gerald Pöltl ◽  
...  
Keyword(s):  
Catalase Activity ◽  
Transient State ◽  
Wild Type ◽  
Deuterium Labeling ◽  
Wild Type Enzyme ◽  
Radical Species ◽  
Distal Side ◽  
In The Wild ◽  
Catalase Peroxidase ◽  
Heme Peroxidases

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp122, Tyr249, and Met275(SynechocysisKatG numbering). The known crystal structures suggest that Tyr249and Met275could be within hydrogen-bonding distance to Arg439. To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg439variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr249and Trp122. In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por·+→ Trp· (or Trp·+) → Tyr·. The M275I variant did not form the Trp· species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp122–Tyr249adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.

The role of distal tryptophan in the bifunctional activity of catalase-peroxidases

2001 ◽  
Vol 29 (2) ◽  
pp. 99-105 ◽  
Author(s):  
G. Regelsberger ◽  
C. Jakopitsch ◽  
P. G. Furtmüller ◽  
F. Rueker ◽  
J. Switala ◽  
...  
Keyword(s):  
Catalase Activity ◽  
Intermediate Compound ◽  
Stopped Flow ◽  
Wild Type ◽  
Compound I ◽  
Wild Type Enzyme ◽  
Catalase Peroxidase ◽  
Electron Reduction ◽  
The One

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

scholarly journals Hydrogen deuterium exchange defines catalytically linked regions of protein flexibility in the catechol O-methyltransferase reaction

2020 ◽  
Vol 117 (20) ◽  
pp. 10797-10805
Author(s):  
Jianyu Zhang ◽  
Jeremy L. Balsbaugh ◽  
Shuaihua Gao ◽  
Natalie G. Ahn ◽  
Judith P. Klinman
Keyword(s):  
Mass Spectrometric Study ◽  
Break Point ◽  
Deuterium Exchange ◽  
Primary Group ◽  
Hydrogen Deuterium Exchange ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
In The Wild ◽  
Donor Acceptor ◽  
Hydrogen Deuterium

Human catechol O-methyltransferase (COMT) has emerged as a model for understanding enzyme-catalyzed methyl transfer from S-adenosylmethionine (AdoMet) to small-molecule catecholate acceptors. Mutation of a single residue (tyrosine 68) behind the methyl-bearing sulfonium of AdoMet was previously shown to impair COMT activity by interfering with methyl donor–acceptor compaction within the activated ground state of the wild type enzyme [J. Zhang, H. J. Kulik, T. J. Martinez, J. P. Klinman, Proc. Natl. Acad. Sci. U.S.A. 112, 7954–7959 (2015)]. This predicts the involvement of spatially defined protein dynamical effects that further tune the donor/acceptor distance and geometry as well as the electrostatics of the reactants. Here, we present a hydrogen/deuterium exchange (HDX)-mass spectrometric study of wild type and mutant COMT, comparing temperature dependences of HDX against corresponding kinetic and cofactor binding parameters. The data show that the impaired Tyr68Ala mutant displays similar breaks in Arrhenius plots of both kinetic and HDX properties that are absent in the wild type enzyme. The spatial resolution of HDX below a break point of 15–20 °C indicates changes in flexibility across ∼40% of the protein structure that is confined primarily to the periphery of the AdoMet binding site. Above 20 °C, Tyr68Ala behaves more like WT in HDX, but its rate and enthalpic barrier remain significantly altered. The impairment of catalysis by Tyr68Ala can be understood in the context of a mutationally induced alteration in protein motions that becomes manifest along and perpendicular to the primary group transfer coordinate.

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe

1999 ◽  
Vol 45 (2) ◽  
pp. 125-129 ◽  
Author(s):  
Norihiro Mutoh ◽  
Chiaki W Nakagawa ◽  
Kenichiro Yamada
Keyword(s):  
Hydrogen Peroxide ◽  
Schizosaccharomyces Pombe ◽  
Catalase Activity ◽  
Parent Strain ◽  
Normal Growth ◽  
Wild Type ◽  
Catalase Gene ◽  
In The Wild ◽  
High Concentrations

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wild-type cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.Key words: catalase, gene disruption, induced hydrogen peroxide resistance, overexpression, Schizosaccharomyces pombe.

scholarly journals Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds

1997 ◽  
Vol 327 (2) ◽  
pp. 537-544 ◽  
Author(s):  
F. Catherine OLIVER ◽  
Sandeep MODI ◽  
U. William PRIMROSE ◽  
Lu-Yun LIAN ◽  
C. K. Gordon ROBERTS
Keyword(s):  
Fatty Acids ◽  
Bacillus Megaterium ◽  
Three Dimensional ◽  
Pair Interaction ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Steady State Kinetics ◽  
In The Wild ◽  
Cytochrome P 450

Oligonucleotide-directed mutagenesis has been used to replace arginine-47 with glutamate in cytochrome P-450 BM3 from Bacillus megaterium and in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measurements of substrate binding. The mutant retains significant catalytic activity towards C12-C16 fatty acids, catalysing hydroxylation in the same (ω-1, ω-2, ω-3) positions with kcat/Km values a factor of 14-21 lower. C12-C16 alkyl trimethylammonium compounds are relatively poor substrates for the wild-type enzyme, but are efficiently hydroxylated by the arginine-47 → glutamate mutant at the ω-1, ω-2 and ω-3 positions, with kcat values of up to 19 s-1. Optical spectroscopy shows that the binding of the C14 and C16 alkyl trimethylammonium compounds to the mutant is similar to that of the corresponding fatty acids to the wild-type enzyme. Paramagnetic relaxation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 Å closer to the iron, than seen in the wild-type, although this difference is much smaller (~ 0.2 Å) in the ferrous state of the complex. The binding of a substrate having the same charge as residue 47 to the ferric state of the enzyme is roughly ten times weaker than that of a substrate having the opposite charge (and thus is able to make an ion-pair interaction with this residue). The results are discussed in the light of the three-dimensional structure of the enzyme.

A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium

1991 ◽  
Vol 69 (4) ◽  
pp. 232-238 ◽  
Author(s):  
Deborah H. Anderson ◽  
Lynda J. Donald ◽  
Mary V. Jacob ◽  
Harry W. Duckworth
Keyword(s):  
Escherichia Coli ◽  
Model Building ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Difference Spectrum ◽  
Site Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
In The Wild

We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine. In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 ± 0.2) to hyperbolic (Hill parameter = 1.0 ± 0.1) and dependence on the activator KCl is greatly reduced. Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described. In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the Arg-319 to Leu mutation did. Small changes in kinetic properties were also obtained with an Arg-319 → Glu (CS R319E) mutation. In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl. To account for these observations we postulate that wild-type E. coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R′, by KCl. In the CS R319L mutant, it is proposed that the T ↔ R equilibrium is shifted significantly towards R state, permitting an easier interaction with substrates in the absence of KCl. To account for the behaviour of enzymes mutated at amino acids 319 and 343, we propose that the allosteric transition between T and R states involves a subtle adjustment of the relative positions of the P and Q helices, which is affected by some of the mutations tested.Key words: citrate synthase, allostery, site-directed mutagenesis.

scholarly journals Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

2021 ◽  
Vol 22 (24) ◽  
pp. 13611
Author(s):  
Juan Antonio Méndez-Líter ◽  
Iván Ayuso-Fernández ◽  
Florian Csarman ◽  
Laura Isabel de Eugenio ◽  
Noa Míguez ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Phylogenetic Analyses ◽  
Carbohydrate Binding ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Lytic Polysaccharide Monooxygenase ◽  
In The Wild ◽  
Type Sequence ◽  
Polysaccharide Monooxygenase

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

scholarly journals Oxidative Stress in Synechococcus sp. Strain PCC 7942: Various Mechanisms for H2O2 Detoxification with Different Physiological Roles

2003 ◽  
Vol 185 (12) ◽  
pp. 3654-3660 ◽  
Author(s):  
Alexander Perelman ◽  
Avraham Uzan ◽  
Dalia Hacohen ◽  
Rakefet Schwarz
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Double Mutant ◽  
Wild Type ◽  
Gene Encoding ◽  
Thioredoxin Peroxidase ◽  
In The Wild ◽  
Catalase Peroxidase ◽  
Pcc 7942 ◽  
Radiation Conditions

ABSTRACT This study focuses on the mechanisms for hydrogen peroxide detoxification in Synechococcus sp. strain PCC 7942. To gain better understanding of the role of different routes of hydrogen peroxide detoxification, we inactivated tplA (thioredoxin-peroxidase-like), which we recently identified. In addition, we inactivated the gene encoding catalase-peroxidase and examined the ability to detoxify H2O2 and to survive oxidative stress in both of the single mutants and in the double mutant. Surprisingly, we observed that the double mutant survived H2O2 concentrations that the single catalase-peroxidase mutant could not tolerate. This phenotype correlated with an increased ability of the double mutant to detoxify externally added H2O2 compared to the catalase-peroxidase mutant. Therefore, our studies suggested the existence of a hydrogen peroxide detoxification activity in addition to catalase-peroxidase and thioredoxin-peroxidase. The rate of detoxification of externally added H2O2 was similar in the wild-type and the TplA mutant cells, suggesting that, under these conditions, catalase-peroxidase activity was essential for this process and TplA was dispensable. However, during excessive radiation, conditions under which the cell might experience oxidative stress, TplA appears to be essential for growth, and cells lacking it cannot compete with the wild-type strain. Overall, these studies suggested different physiological roles for various cellular hydrogen peroxide detoxification mechanisms in Synechococcus sp. strain PCC 7942.

scholarly journals Salinity Stress-Mediated Suppression of Expression of Salt Overly Sensitive Signaling Pathway Genes Suggests Negative Regulation by AtbZIP62 Transcription Factor in Arabidopsis thaliana

2020 ◽  
Vol 21 (5) ◽  
pp. 1726 ◽  
Author(s):  
Nkulu Kabange Rolly ◽  
Qari Muhammad Imran ◽  
In-Jung Lee ◽  
Byung-Wook Yun
Keyword(s):  
Arabidopsis Thaliana ◽  
Signaling Pathway ◽  
Salinity Stress ◽  
Catalase Activity ◽  
Antioxidant Systems ◽  
Wild Type ◽  
Pheophytin A ◽  
Sos Pathway ◽  
In The Wild ◽  
Salt Overly Sensitive

Salt stress is one of the most serious threats in plants, reducing crop yield and production. The salt overly sensitive (SOS) pathway in plants is a salt-responsive pathway that acts as a janitor of the cell to sweep out Na+ ions. Transcription factors (TFs) are key regulators of expression and/or repression of genes. The basic leucine zipper (bZIP) TF is a large family of TFs regulating various cellular processes in plants. In the current study, we investigated the role of the Arabidopsis thaliana bZIP62 TF in the regulation of SOS signaling pathway by measuring the transcript accumulation of its key genes such as SOS1, 2, and 3, in both wild-type (WT) and atbzip62 knock-out (KO) mutants under salinity stress. We further observed the activation of enzymatic and non-enzymatic antioxidant systems in the wild-type, atbzip62, atcat2 (lacking catalase activity), and atnced3 (lacking 9-cis-epoxycarotenoid dioxygenase involved in the ABA pathway) KO mutants. Our findings revealed that atbzip62 plants exhibited an enhanced salt-sensitive phenotypic response similar to atnced3 and atcat2 compared to WT, 10 days after 150 mM NaCl treatment. Interestingly, the transcriptional levels of SOS1, SOS2, and SOS3 increased significantly over time in the atbzip62 upon NaCl application, while they were downregulated in the wild type. We also measured chlorophyll a and b, pheophytin a and b, total pheophytin, and total carotenoids. We observed that the atbzip62 exhibited an increase in chlorophyll and total carotenoid contents, as well as proline contents, while it exhibited a non-significant increase in catalase activity. Our results suggest that AtbZIP62 negatively regulates the transcriptional events of SOS pathway genes, AtbZIP18 and AtbZIP69 while modulating the antioxidant response to salt tolerance in Arabidopsis.

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