High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases

Zámocký, Marcel; García-Fernández, Queralt; Gasselhuber, Bernhard; Jakopitsch, Christa; Furtmüller, Paul G.; Loewen, P.C.; Fita, Ignacio; Obinger, Christian; Carpena, X.

doi:10.1074/jbc.m112.384271

Cited by 24 publications

(18 citation statements)

References 34 publications

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“…The crystal structures of KatGs from Haloarcula marismortui (HmKatG; Yamada et al, 2002), Burkholderia pseudomallei (BpKatG; Carpena et al, 2003), Mycobacterium tuberculosis (MtKatG; Bertrand et al, 2004) and Magnaporthe grisea (MagKatG;Zá mocký et al, 2012) have been reported. The X-ray structures revealed that KatGs possess unique covalent bonds formed among the side chains of three distal residues, Met-Tyr-Trp, which are located on the distal side of the haem active site.…”

Section: Introductionmentioning

confidence: 99%

The 2.2 Å resolution structure of the catalase-peroxidase KatG fromSynechococcus elongatusPCC7942

Kamachi¹,

Wada²,

Tamoi³

et al. 2014

Acta Cryst Sect F

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The crystal structure of catalase-peroxidase from Synechococcus elongatus PCC7942 (SeKatG) was solved by molecular replacement and refined to an Rwork of 16.8% and an Rfree of 20.6% at 2.2 Å resolution. The asymmetric unit consisted of only one subunit of the catalase-peroxidase molecule, including a protoporphyrin IX haem moiety and two sodium ions. A typical KatG covalent adduct was formed, Met248-Tyr222-Trp94, which is a key structural element for catalase activity. The crystallographic equivalent subunit was created by a twofold symmetry operation to form the functional dimer. The overall structure of the dimer was quite similar to other KatGs. One sodium ion was located close to the proximal Trp314. The location and configuration of the proximal cation site were very similar to those of typical peroxidases such as ascorbate peroxidase. These features may provide a structural basis for the behaviour of the radical localization/delocalization during the course of the enzymatic reaction.

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Section: Introductionmentioning

confidence: 99%

The 2.2 Å resolution structure of the catalase-peroxidase KatG fromSynechococcus elongatusPCC7942

Kamachi¹,

Wada²,

Tamoi³

et al. 2014

Acta Cryst Sect F

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“…LL1 bears the strictly conserved tyrosine (Y226 by E. coli KatG numbering) which participates in a unique MYW covalent adduct found on the distal side of the heme. 8,18,22,23,[29][30][31][32] Replacement of any of the members of the adduct invariably eliminates catalase activity without appreciably diminishing the peroxidatic capabilities of the enzyme. 30,[32][33][34][35][36][37][38][39] 47 Finally, variants which delete the C-terminal end of LL1 not only eliminate catalase activity as expected, they also produce substantial gains in peroxidatic activity even over other catalase-negative variants (e.g., Y226F), indicating that LL1 may favor catalase over peroxidase activity by also restricting access of electron donors to the heme edge.…”

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confidence: 99%

A Role for Catalase-Peroxidase Large Loop 2 Revealed by Deletion Mutagenesis: Control of Active Site Water and Ferric Enzyme Reactivity

Kudalkar

Njuma

et al. 2015

Biochemistry

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Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

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“…Recently, the first structures of a eukaryotic bifunctional catalase-peroxidase (KatGs) in the ferric resting 1 and oxoiron 2 state were published. Similar to the prokaryotic counterparts, 3 − 7 eukaryotic KatGs have two interdependent cooperating redox-active cofactors at their active site, namely a heme b and a unique posttranslationally and autocatalytically formed Met299-Tyr273-Trp140 (MYW) adduct [extracellular Magnaporthe grisea KatG ( Mag KatG2) numbering throughout].…”

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confidence: 99%

“… 9 , 10 Together with cytochrome c peroxidases, ascorbate peroxidases and hybrid-type peroxidases KatGs comprise Family I of the peroxidase-catalase superfamily. 11 , 12 Besides the MYW adduct mentioned above, further KatG-specific structural peculiarities include (i) a two-domain structure with only the N-terminal domain containing the two redox cofactors and essential catalytic residues, 1 , 3 , 4 (ii) loop insertions that contribute to the architecture of the long and restricted access channel, and (iii) a fully conserved aspartate at the entrance of the substrate channel to the heme cavity. 13 , 14 Mutation of any of these KatG-typical structural features typically reduces the catalase activity without compromising the peroxidase activity.…”

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confidence: 99%

“…Although several years of elaborate research have passed, there are still open questions about the catalatic reaction of KatGs. The recent finding that eukaryotic Mag KatG2 has some novel structural features 1 , 2 , 35 and a more acidic pH optimum (i.e., pH 5.25) for H 2 O 2 dismutation 36 now allows us to scrutinize whether the postulated catalatic mechanism ( reactions I – IV ) and the role of the mobile arginine in modulating the pH dependence are also valid for eukaryotic KatGs. In this work, we have determined the X-ray structures of Mag KatG2 at pH 3, 5.5, and 7.0 and investigated the dynamics of Arg461 in ferric Mag KatG2 by molecular dynamics simulation.…”

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confidence: 99%

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Interaction with the Redox Cofactor MYW and Functional Role of a Mobile Arginine in Eukaryotic Catalase-Peroxidase

et al. 2016

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Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases with an additional posttranslationally formed redox-active Met-Tyr-Trp cofactor that is essential for catalase activity. On the basis of studies of bacterial KatGs, controversial mechanisms of hydrogen peroxide oxidation were proposed. The recent discovery of eukaryotic KatGs with differing pH optima of catalase activity now allows us to scrutinize those postulated reaction mechanisms. In our study, secreted KatG from the fungus Magnaporthe grisea (MagKatG2) was used to analyze the role of a remote KatG-typical mobile arginine that was shown to interact with the Met-Tyr-Trp adduct in a pH-dependent manner in bacterial KatGs. Here we present crystal structures of MagKatG2 at pH 3.0, 5.5, and 7.0 and investigate the mobility of Arg461 by molecular dynamics simulation. Data suggest that at pH ≥4.5 Arg461 mostly interacts with the deprotonated adduct Tyr. Elimination of Arg461 by mutation to Ala slightly increases the thermal stability but does not alter the active site architecture or the kinetics of cyanide binding. However, the variant Arg461Ala lost the wild-type-typical optimum of catalase activity at pH 5.25 (kcat = 6450 s–1) but exhibits a broad plateau between pH 4.5 and 7.5 (kcat = 270 s–1 at pH 5.5). Moreover, significant differences in the kinetics of interconversion of redox intermediates of wild-type and mutant protein mixed with either peroxyacetic acid or hydrogen peroxide are observed. These findings together with published data from bacterial KatGs allow us to propose a role of Arg461 in the H2O2 oxidation reaction of KatG.

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High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases

Cited by 24 publications

References 34 publications

The 2.2 Å resolution structure of the catalase-peroxidase KatG fromSynechococcus elongatusPCC7942

The 2.2 Å resolution structure of the catalase-peroxidase KatG fromSynechococcus elongatusPCC7942

A Role for Catalase-Peroxidase Large Loop 2 Revealed by Deletion Mutagenesis: Control of Active Site Water and Ferric Enzyme Reactivity

Interaction with the Redox Cofactor MYW and Functional Role of a Mobile Arginine in Eukaryotic Catalase-Peroxidase

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