Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Singh, Rahul; Wiseman, B.; Deemagarn, Taweewat; Donald, Lynda J.; Duckworth, Harry W.; Carpena, X.; Fita, Ignacio; Loewen, P.C.

doi:10.1074/jbc.m406374200

Cited by 81 publications

(93 citation statements)

References 42 publications

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“…Our findings that the oxidase activity exhibited unique substrate and cofactor requirements, as well as reaction mechanisms, demonstrate that catalase oxidase activity is distinct from its previously recognized catalatic and peroxidatic activities. In this regard, the new enzymatic activity, which was oxygen-dependent, did not require the addition of hydrogen peroxide or any additional cofactors, similar to the oxidase activity found in catalase-peroxidase KatG (33). In addition, unlike the catalatic activity of catalase, which does not strictly follow classic Michaelis-Menten kinetics, presumably due to the high reaction rate and inactivation of the enzyme at high hydrogen peroxide concentrations (13,34), catalase oxidase activity was readily saturable and reversible.…”

Section: Discussionmentioning

confidence: 72%

Characterization of the Oxidase Activity in Mammalian Catalase

Vetrano

Heck

Mariano³

et al. 2005

Journal of Biological Chemistry

139

101

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Catalase is a highly conserved heme-containing antioxidant enzyme known for its ability to degrade hydrogen peroxide into water and oxygen. In low concentrations of hydrogen peroxide, the enzyme also exhibits peroxidase activity. We report that mammalian catalase also possesses oxidase activity. This activity, which is detected in purified catalases, cell lysates, and intact cells, requires oxygen and utilizes electron donor substrates in the absence of hydrogen peroxide or any added cofactors. Using purified bovine catalase and 10-acetyl-3,7-dihydroxyphenoxazine as the substrate, the oxidase activity was found to be temperature-dependent and displays a pH optimum of 7-9. The K m for the substrate is 2. Mammalian catalase belongs to a family of Fe-protoporphyrin IX containing proteins that include a variety of cytochromes, globins, and peroxidases and is one of the best characterized antioxidant enzymes (1). As a homotetrameric heme-containing enzyme, it is known for its ability to convert hydrogen peroxide into water and oxygen (catalatic activity), and in the presence of low concentrations of hydrogen peroxide to oxidize low molecular weight alcohols (peroxidatic activity). The conversion of hydrogen peroxide to water and oxygen by catalase is a two-step process whereby catalase heme Fe 3ϩ reduces one molecule of hydrogen peroxide to water, generating a covalent Fe 4ϩ ϭO oxyferryl species and a porphyrin cation radical. This reaction intermediate, referred to as compound I, then oxidizes a second hydrogen peroxide molecule forming molecular oxygen and water (1-3) (see Fig. 1). The peroxidatic activity of catalase results from the ability of compound I to oxidize alcohols to aldehydes and water (4 -6) ( Fig. 1). Each catalase monomer binds one molecule of heme; the holoenzyme also binds two molecules of NADPH, although the precise role of this cofactor in enzymatic activity is unclear, because hydrogen peroxide provides both oxidative and reductive potential during catalysis. Recent studies suggest that NADPH may be important in maintaining catalase in an active state (7).In mammalian cells, catalase is found at high concentrations in peroxisomes, along with a variety of oxidases and peroxidases (8). It has been suggested that the enzyme protects cells by removing hydrogen peroxide produced by flavin containing oxidases in the peroxisome, thereby preventing the accumulation of toxic levels of this reactive oxygen intermediate (9). However, hydrogen peroxide is important for an array of activities, including peroxidase-mediated metabolism, in cells, and potentially, without this reactive oxygen intermediate, cellular functioning would be limited. In addition, the K m for the catalatic activity of catalase is Ͼ10 mM, therefore, at low intracellular concentrations of hydrogen peroxide, this reaction is not kinetically favored, and it is assumed that peroxidases such as glutathione peroxidase or the recently discovered l-Cys peroxiredoxins effectively lower intracellular concentrations of hydrogen peroxide (10). In...

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Section: Discussionmentioning

confidence: 72%

Characterization of the Oxidase Activity in Mammalian Catalase

Vetrano

Heck

Mariano³

et al. 2005

Journal of Biological Chemistry

139

101

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“…A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9, 10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4, 15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.…”

Section: Isonicotinic Acid Hydrazide (Isoniazid or Inh)mentioning

confidence: 99%

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

Wiseman

Carpena

Feliz

et al. 2010

Journal of Biological Chemistry

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Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis Isonicotinic acid hydrazide (isoniazid or INH)3 is a widely used anti-tubercular pro-drug that requires activation in a reaction involving the catalase-peroxidase KatG of Mycobacterium tuberculosis (MtKatG) (1) whereby the hydrazine group is removed and the isonicotinyl portion is added to NAD ϩ to generate isonicotinyl-NAD or IN⅐NAD (see Fig. 1). Once formed, IN⅐NAD inhibits the synthesis of mycolic acids, and therefore, the growth of M. tuberculosis, by binding to the longchain enoyl acyl carrier protein reductase (InhA) (2). Despite understanding the role of IN⅐NAD in the inhibition of mycolic acid synthesis and knowing that KatG is required for INH activation in vivo, uncertainties about the mechanism of its formation remain.The involvement of MtKatG in INH activation suggested that the peroxidatic process had a role, and this provided a focus for several studies employing external oxidants such as peroxyacetic acid (3), t-butyl hydroperoxide (4 -6) and low levels of H 2 O 2 (7, 6) to activate the peroxidatic pathway for INH oxidation and activation. In addition, EPR studies have demonstrated that INH can serve as an electron source to reduce peroxidatic intermediates, including specific Trp ⅐ radical species following peroxyacetic acid oxidation of MtKatG (3,8).A multiplicity of methods has been employed to directly and indirectly assay INH activation, including the determination of INH oxidation to isonicotinic acid (9, 10), the HPLC assay of INH disappearance (11), the inactivation of InhA in a mixture of InhA and KatG (7,12,13,14), the HPLC detection of IN⅐NAD (4, 15), and the direct measurement of IN⅐NAD using its characteristic absorbance at 326 nm (6,7,12,15,16). Reports of INH activation in mixtures lacking an external oxidant (4,5,6,9,12,14,15) initially suggested that the peroxidatic process may not be required, but the mixtures of INH, NADH, and KatG would have supported NADH reduction of molecular oxygen to superoxide and low levels of H 2 O 2 (15) to activate the peroxidase reaction.Despite the considerable evidence that a peroxidatic process is involved in IN⅐NAD synthesis, attempts to rationalize the reaction entirely in terms of the peroxidatic pathway generally produced models that were incomplete from the standpoint of electron balance (5,7,10) or that involved hypothetical intermediates not characterized in any other system (5, 6). For example, a common scheme shows the isonicotinyl radical, generated in a peroxidatic reaction, reacting with NAD ϩ to yield IN⅐NAD, when such a reaction would actually yield the IN⅐NAD ϩ ⅐ radical (Fig. 1). The need to reduce this radical in an oxidizing environment suggests that more than a simple peroxidatic pathway is involved in the INH activation process. This rationale leads to superoxide, O 2. , a known reducing agent with

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“…It is possible that in the pH ranges of ¡7.0 and .7.0, two different sets of amino acid residues of the protein play critical roles in catalysis. Another explanation is that at pH .7.0, rMjNox produces superoxide; certain Fe-containing Nox enzymes, which do not belong to the FDR family, exhibit such a behaviour (Singh et al, 2004). rMjNox did produce superoxide and the activity was elevated at pH .8.5.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

2009

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Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit) "1 , showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit) "1 and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were ¢10 and ¢95 6C, respectively. At pH 7 and 83 6C, apparent K m values for NADH and O 2 were 3 mM and 1.9 mM, respectively, and the specific activity at 1.4 mM O 2 was 60 mmol min "1 mg "1 ; 62 % of NADH-derived reducing equivalents were recovered as H 2 O 2 and the rest probably generated H 2 O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,59-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high K m , O 2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

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Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Cited by 81 publications

References 42 publications

Characterization of the Oxidase Activity in Mammalian Catalase

Characterization of the Oxidase Activity in Mammalian Catalase

Isonicotinic Acid Hydrazide Conversion to Isonicotinyl-NAD by Catalase-peroxidases

Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

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