Total Conversion of Bifunctional Catalase-Peroxidase (KatG) to Monofunctional Peroxidase by Exchange of a Conserved Distal Side Tyrosine

Jakopitsch, Christa; Auer, Markus; Ivancich, Anabella; Rüker, Florian; Furtmüller, Paul G.; Obinger, Christian

doi:10.1074/jbc.m211625200

Cited by 77 publications

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“…Upon exchange of Tyr 249 by phenylalanine the enzyme was totally converted from a bifunctional peroxidase to a monofunctional peroxidase (11). Exchange of both Met 275 and Arg 439 also had a dramatic influence on the catalatic activity ( Table I).…”

Section: Resultsmentioning

confidence: 91%

“…Cloning, expression, and purification of wild-type KatG and the M275I and the Y249F variants from Synechocystis were described previously (11,7,14). Isotope labeling and purification of specifically deuterated KatGs were described recently by Ivancich et al (13).…”

Section: Methodsmentioning

confidence: 99%

“…Upon exchange of the distal Trp (Trp 122 in Synechocystis) the catalase activity was dramatically diminished in the Escherichia coli (9) or even lost in the Synechocystis (10) enzymes, whereas the peroxidase activity was not affected in both cases. 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).…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Jakopitsch¹,

Ivancich

Schmuckenschlager³

et al. 2004

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

confidence: 91%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Jakopitsch¹,

Ivancich

Schmuckenschlager³

et al. 2004

Journal of Biological Chemistry

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“…This M-Y-W crosslink appears to be a characteristic common to all KatGs and has been demonstrated to be essential for the catalase activity [9,15,16,19]. Interestingly, this adduct can be associated with a KatG-typical arginine (R439) [2].…”

Section: Resultsmentioning

confidence: 96%

Hydrogen peroxide oxidation by catalase‐peroxidase follows a non‐scrambling mechanism

et al. 2007

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Indole N‐Linked Hydroperoxyl Adduct of Protein‐Derived Cofactor Modulating Catalase‐Peroxidase Functions

Li,

Duan,

Traore

et al. 2024

Angewandte Chemie

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Bifunctional catalase‐peroxidase (KatG) features a posttranslational methionine‐tyrosine‐tryptophan (MYW) crosslinked cofactor crucial for its catalase function, enabling pathogens to neutralize hydrogen peroxide during infection. We discovered the presence of indole nitrogen‐linked hydroperoxyl adduct (MYW‐OOH) in Mycobacterium tuberculosis KatG in the solution state under ambient conditions, suggesting its natural occurrence. By isolating predominantly MYW‐OOH‐containing KatG protein, we investigated the chemical stability and functional impact of MYW‐OOH. We discovered that MYW‐OOH inhibits catalase activity, presenting a unique temporary lock. Exposure to peroxide or increased temperature removes the hydroperoxyl adduct from the protein cofactor, converting MYW‐OOH to MYW and restoring the detoxifying ability of the enzyme against hydrogen peroxide. Thus, the N‐linked hydroperoxyl group is releasable. KatG with MYW‐OOH represents a catalase dormant, but primed, state of the enzyme. These findings provide insight into chemical strategies targeting the bifunctional enzyme KatG in pathogens, highlighting the role of N‐linked hydroperoxyl modifications in enzymatic function.

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Total Conversion of Bifunctional Catalase-Peroxidase (KatG) to Monofunctional Peroxidase by Exchange of a Conserved Distal Side Tyrosine

Abstract: ؊1 at pH 7 and 15°C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure.

Cited by 77 publications

References 29 publications

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

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

Hydrogen peroxide oxidation by catalase‐peroxidase follows a non‐scrambling mechanism

Indole N‐Linked Hydroperoxyl Adduct of Protein‐Derived Cofactor Modulating Catalase‐Peroxidase Functions

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