Abstract
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 microm), 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.
Highlights
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 central role of KatG in isonicotinic acid hydrazide (INH) activation is evident in the significant fraction of INH-resistant cases of tuberculosis attributable to mutations in katG and in biochemical studies that have demonstrated a direct role for KatG in the generation of various isonicotinoyl derivatives [6]
The absence of H2O2 involvement in INH activation implies that a reaction different from either the peroxidase or the catalase reactions is involved, and some reports have suggested that the active participation of KatG in INH activation involves more than just hydrazinolysis [9]
Summary
Strains and Plasmids—The plasmids pAH1 [23], pBpKatG [24], and pBT22 [25] were used as the source of catalase-peroxidases from M. tuberculosis, Burkholderia pseudomallei, and Escherichia coli, respectively. One unit of peroxidase is defined as the amount that decomposes 1 mol of ABTS in 1 min in a solution of 0.3 mM ABTS (⑀ ϭ 36,800 MϪ1 cmϪ1) and 2.5 mM H2O2 at pH 4.5 and 25 °C. Gel electrophoresis was carried out under nondenaturing conditions according to Davis [32], except in pH 8.1 Tris-HCl. Following electrophoresis, peroxidase activity was visualized by the method of Gregory and Fridovich [33], and catalase was visualized as described by Clare et al [34], but using 20 mM H2O2 for better contrast. The sequence alignment file included 52 catalase-peroxidase sequences and the structure file for B. pseudomallei KatG (Protein Data Bank code 1MWV) was used as the model. The source code or IRIX 6.5 executable for CONRES can be obtained from the authors on request
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