The 2.0 Å crystal structure of catalase-peroxidase from Haloarcula marismortui

The heme-containing catalase-peroxidases are bifunctional enzymes that degrade hydrogen peroxide either as a catalaseThe catalatic reaction, with a more rapid turnover rate, dominates over the peroxidatic reaction, and the in vivo peroxidatic substrate remains unidentified, suggesting that the main role of the enzyme is the removal of H 2 O 2 , preventing the formation of highly reactive and damaging breakdown products of H 2 O 2 . However, the enzyme has a close sequence resemblance to plant peroxidases (1, 2), and it remains a possibility that the peroxidatic reaction has a metabolic significance outside of degrading H 2 O 2 . Indeed, it is clear that the catalatic function evolved as an adaptation of the peroxidatic function because the simple change of a tryptophan to a phenylalanine in the distal heme pocket reduces catalatic activity by 1000-fold (of Escherichia coli HPI) and increases peroxidatic activity by 3-fold (3-5). Furthermore, the core structures of both the N-and C-terminal domains of the catalase-peroxidases from Haloarcula marismortui and Burkholderia pseudomallei closely resemble the structure of plant peroxidases (6, 7). Finally, the conversion of isoniazid into its active antitubercular form by KatG of Mycobacterium tuberculosis is clearly a result of the peroxidatic reaction using isonicotinic acid hydrazide (INH) 1 as a substrate that must mimic the actual in vivo substrate.The structures of the catalase-peroxidases from H. marismortui and B. pseudomallei have been reported (6, 7) and have revealed several features that are, so far, unique to this class of enzyme. Present in both structures is an unusual adduct or covalent linkage among the side chains of a tryptophan, a tyrosine, and a methionine (see Fig. 1). The likely mechanistic significance of the covalent structure is enhanced by the fact that the tryptophan lies in the active site and is essential for catalatic activity. A second feature, evident only in the structure of BpKatG, is a modification to the heme, likely a hydroperoxide group added to ring I of the heme in close proximity to the Trp-Tyr-Met adduct. This paper presents mass spectrometry evidence supportive of the existence of the TrpTyr-Met adduct originally deduced from the electron density maps of HmCPx and BpKatG (6, 7). EXPERIMENTAL PROCEDURESMaterials-Standard chemicals and biochemicals were obtained from Sigma. Restriction endonucleases, polynucleotide kinase, DNA ligase, and the Klenow fragment of DNA polymerase were obtained from Invitrogen.Strains and Plasmids-The plasmid pBG306 encoding BpKatG (8) and the plasmids pAH8 and pW105F encoding KatG (HPI) and its W105F variant from E. coli (3) were transformed into strain UM262 pro leu rpsL hsdM hsdR endI lacY katG2 katE12::Tn10 recA (8) for expression of the katG constructs and isolation of the mutant KatG proteins. Phagemids pKS ϩ and pKS Ϫ from Stratagene Cloning Systems were used for mutagenesis, sequencing, and cloning. E. coli strains NM522 (supE thi) (lac-proAB) hsd-5 [FЈ proAB lacI q lacZ)15]) (9), JM109 (r...

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confidence: 99%

“…The structures of the catalase-peroxidases from H. marismortui and B. pseudomallei have been reported (6,7) and have revealed several features that are, so far, unique to this class of enzyme. Present in both structures is an unusual adduct or covalent linkage among the side chains of a tryptophan, a tyrosine, and a methionine (see Fig.…”

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confidence: 99%

Characterization of the Catalase-Peroxidase KatG from Burkholderia pseudomallei by Mass Spectrometry

Donald

Krokhin

Duckworth

et al. 2003

“…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 arginine, histidine, tryptophan, and the proximal triad aspartate, tryptophan, and histidine.…”

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confidence: 99%

“…An unprecedented covalent bond between the distal side tryptophan, tyrosine, and methionine residues (in Synechocystis numbering Trp 122 , Tyr 249 , and Met 275 ) was proposed to be present in the H. marismortui enzyme (5). A mass spectrometric analysis of the tryptic peptides from recombinant wild-type KatG and the tryptophan, tyrosine, and methionine variants of Synechocystis (7) and B. pseudomallei (8) confirmed that this novel covalent adduct really exists in solution and thus may be a common feature to all catalase-peroxidases.…”

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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...

“…the superfamily of peroxidases from archaea, bacteria, fungi, and plants and the superfamily of animal peroxidases). Despite the availability of crystal structures (1)(2)(3)(4), no structural bases for the high catalase activity of KatGs has been assigned so far. Whether in KatGs compound I plays a similar role in the catalase cycle as in monofunctional catalases (EC 1.11.1.6; see (5) is not clear at the moment.…”

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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