Rapid Formation of Compound II and a Tyrosyl Radical in the Y229F Mutant of Mycobacterium tuberculosis Catalase-peroxidase Disrupts Catalase but Not Peroxidase Function

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD ؉ and either H 2 O 2 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 (K m ‫؍‬ 12 M), but the oxidase reaction is slow (k cat ‫؍‬ 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 H 2 O 2 -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.

Section: Nadh Oxidation By Katg-nadsupporting

confidence: 79%

Catalase-peroxidases (KatG) Exhibit NADH Oxidase Activity

Singh

Wiseman

Deemagarn

et al. 2004

“…Most interestingly, disruption of this covalent link by exchange of these tyrosines converted the bifunctional KatGs to typical monofunctional heme peroxidases. The catalase activity was dramatically decreased, the formation of (conventional) compound I could be followed even with equimolar H 2 O 2 , and a typical (ferryl-)compound II was formed, which was easily transformed to compound III (13,14).…”

mentioning

confidence: 99%

“…1, Reaction 10) (12) nor the transition of compound II to compound III has been investigated so far, although the knowledge of these reactions could help to understand the KatG-specific high catalase activity. This is most obvious when looking at the variants Y249F of Synechocystis KatG (13) or Y229F of Mycobacterium KatG (14). These tyrosines are part of a KatG-specific covalent link at the distal heme side, formed among the side chains of conserved tryptophan, tyrosine and methionine.…”

mentioning

confidence: 99%

Kinetics of Interconversion of Ferrous Enzymes, Compound II and Compound III, of Wild-type Synechocystis Catalase-peroxidase and Y249F

Jakopitsch

Wanasinghe

Jantschko

et al. 2005

“…Moreover, upon exchange of the cross-linked Tyr residue (Tyr 249 in Synechocystis) the bifunctional enzyme was totally converted to a monofunctional peroxidase (11). The Y249F variant shows spectral and kinetic features characteristic of most plant-type peroxidase intermediates, namely compounds I and II (11,12), thus different from the wild-type KatG (10,11). Additionally, the B. pseudomallei variant M264L (Met 275 in Synechocystis) showed a significantly reduced overall catalase but an intact peroxidase activity (8).…”

mentioning

confidence: 99%

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

Jakopitsch¹,

Ivancich

Schmuckenschlager³

et al. 2004

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 Trp 122 , Tyr 249 , and Met 275 (Synechocysis KatG numbering). The known crystal structures suggest that Tyr 249 and Met 275 could be within hydrogen-bonding distance to Arg 439 . 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 Arg 439 variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr 249 and Trp 122 . 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 oxoferrylporphyrin 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 ⅐ ؉ 3 Trp ⅐ (or Trp ⅐ ؉ ) 3 Tyr ⅐ . The M275I variant did not form the Trp ⅐ species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp 122 -Tyr 249 adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.Catalase-peroxidases (KatGs) 1 are present in bacteria and fungi and function primarily as hydrogen peroxide scavengers (1-3). Sequence similarities indicate they are members of the class I of the superfamily of plant, fungal, and bacterial peroxidases (4). In contrast to the other members of this group, such as cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), they show a high catalase activity comparable with monofunctional catalases. Although the catalase activity is overwhelming, KatGs also show a substantial peroxidase activity with various one electron donors.There are three crystal structures of KatGs available, namely the Archaebacterium Haloarcula marismortui (5), Burkholderia pseudomallei (6), and Synechococcus PCC 7942 (PDB entry 1UB2), the latter being a cyanobacterial KatG with high homology to Synechocystis KatG. The structures show that the arrangement of the active site is similar to CcP and APX, containing the distal triad arg...