Ben Wiseman

Comparative study of catalase-peroxidases (KatGs).

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 15,900s(-1) and 8-25s(-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 IC50 values of 0.2-20muM and 50-170muM, respectively. The K(M)s of the enzymes for H2O2 in the catalase reaction were relatively invariant between 3 and 5mM at pH 7.0, but increased to values ranging from 20 to 225mM at pH 5, consistent with protonation of the distal histidine (pKa approximately 6.2) interfering with H2O2 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 0.002s(-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 (KatG) Exhibit NADH Oxidase Activity

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km = 12 μm), but the oxidase reaction is slow (kcat = 0.54 min-1) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.

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

Activation of the pro-drug isoniazid (INH) as an anti-tubercular drug in Mycobacterium tuberculosis involves its conversion to isonicotinyl-NAD, a reaction that requires the catalase-peroxidase KatG. This report shows that the reaction proceeds in the absence of KatG at a slow rate in a mixture of INH, NAD+, Mn2+, and O2, and that the inclusion of KatG increases the rate by >7 times. Superoxide, generated by either Mn2+- or KatG-catalyzed reduction of O2, is an essential intermediate in the reaction. Elimination of the peroxidatic process by mutation slows the rate of reaction by 60% revealing that the peroxidatic process enhances, but is not essential for isonicotinyl-NAD formation. The isonicotinyl-NAD•+ radical is identified as a reaction intermediate, and its reduction by superoxide is proposed. Binding sites for INH and its co-substrate, NAD+, are identified for the first time in crystal complexes of Burkholderia pseudomallei catalase-peroxidase with INH and NAD+ grown by co-crystallization. The best defined INH binding sites were identified, one in each subunit, on the opposite side of the protein from the entrance to the heme cavity in a funnel-shaped channel. The NAD+ binding site is ∼20 Å from the entrance to the heme cavity and involves interactions primarily with the AMP portion of the molecule in agreement with the NMR saturation transfer difference results.

Distinct role of specific tryptophans in facilitating electron transfer or as [Fe(IV)=O Trp(*)] intermediates in the peroxidase reaction of Bulkholderia pseudomallei catalase-peroxidase: a multifrequency EPR spectroscopy investigation.

We have characterized the reactive intermediates of the peroxidase-like reaction of Bulkholderia pseudomallei KatG using multifrequency EPR spectroscopy. The aim was to investigate the putative role of tryptophanyl radicals as alternative intermediates to the [Fe(IV)=O Por(*+)] species or as short-lived species involved in superexchange-coupled pathways between redox cofactors. Three distinct sites for the formation of radical intermediates, Trp330, Trp139 and Trp153, were identified using single, double and triple variants of Bulkholderia pseudomallei KatG. The proximal Trp330 is the site for a radical in magnetic interaction with the ferryl heme iron [Fe(IV)=O Trp(*+)], formed at the expense of a short-lived [Fe(IV)=O Por(*+)] species as in the cases of Mycobacterium tuberculosis KatG and cytochrome c peroxidase. Formation of the Trp153 radical at a site close to the enzyme surface crucially depends on the integrity of the H-bonding network of the heme distal side, that includes Trp95, the radical site in the Synechocystis KatG. Accordingly, the extended H-bonding network and Trp94 provide an electron transfer pathway between Trp153 and the heme. The distal tryptophan (Trp111) being part of the KatG-specific adduct required for the catalase-like activity, is involved in facilitating electron transfer for the formation of the Trp139 radical. We propose a comprehensive description of the role of specific Trp residues that takes into account not only the apparent differences in sites for the Trp(*) intermediates in other catalase-peroxidases but also the similar cases observed in monofunctional peroxidases.

A molecular switch and electronic circuit modulate catalase activity in catalase‐peroxidases

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.

Two alternative substrate paths for compound I formation and reduction in catalase-peroxidase KatG from Burkholderia pseudomallei.

Five residues in the multifunctional catalase–peroxidase KatG of Burkholderia pesudomallei are essential for catalase, but not peroxidase, activity. Asp141 is the only one of these catalase‐specific residues not related with the covalent adduct found in KatGs that when replaced with a nonacidic residue reduces catalase activity to 5% of native levels. Replacing the nearby catalytic residue Arg108 causes a reduction in catalase activity to 35% of native levels, whereas a variant with both Asp141 and Arg108 replaced exhibits near normal catalase activity (82% of native), suggesting a synergism in the roles of the two residues in support of catalase activity in the enzyme. Among the Asp141 variants, D141E is unique in retaining normal catalase activity but with modified kinetics, suggesting more favorable compound I formation and less favorable compound I reduction. The crystal structure of the D141E variant has been determined at 1.8‐Å resolution, revealing that the carboxylate of Glu141 is moved only slightly compared with Asp141, but retains its hydrogen bond interaction with the main chain nitrogen of Ile237. In contrast, the low temperature ferric Electron Paramagnetic Resonance spectra of the D141A, R108A, and R108A/D141A variants are consistent with modifications of the water matrix and/or the relative positioning of the distal residue side chains. Such changes explain the reduction in catalase activity in all but the double variant R108A/D141A. Two pathways of hydrogen bonded solvent lead from the entrance channel into the heme active site, one running between Asp141 and Arg108 and the second between Asp141 and the main chain atoms of residues 237–239. It is proposed that binding of substrate H2O2 to Asp141 and Arg108 controls H2O2 access to the heme active site, thereby modulating the catalase reaction. Proteins 2007.

Spectroscopic and kinetic investigation of the reactions of peroxyacetic acid with Burkholderia pseudomallei catalase-peroxidase, KatG.

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 [Fe(IV)═O Trp330(•+)], [Fe(IV)═O Trp139(•)], and [Fe(IV)═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 [Fe(IV)═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 W153), 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 [Fe(IV)═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.