Marcel Zámocký

The molecular peculiarities of catalase‐peroxidases

In developing ideas of how protein structure modifies haem reactivity, the activity of Class I of the plant peroxidase superfamily (including cytochrome c peroxidase, ascorbate peroxidase and catalase‐peroxidases (KatGs)) is an exciting field of research. Despite striking sequence homologies, there are dramatic differences in catalytic activity and substrate specificity with KatGs being the only member with substantial catalase activity. Based on multiple sequence alignment performed for Class I peroxidases, we present a hypothesis for the pronounced catalase activity of KatGs. In their catalytic domains KatGs are shown to possess three large insertions, two of them are typical for KatGs showing highly conserved sequence patterns. Besides an extra C‐terminal copy of the ancestral hydroperoxidase gene resulting from gene duplication, these two large loops are likely to control the orientation of both the haem group and of essential residues in the active site. They seem to modulate the access of substrates to the prosthetic group at the distal side as well as the flexibility and character of the bond between the proximal histidine and the ferric iron. The hypothesis presented opens new possibilities in the rational engineering of peroxidases.

Molecular evolution of hydrogen peroxide degrading enzymes.

Graphical abstract Highlights ► Detailed molecular evolution of metalloenzymes that catalyse the dismutation of hydrogen peroxide. ► Three protein families of differing structure, catalytic mechanism, distribution and evolutionary age. ► Catalatic enzymes in pathogenic organisms are promising targets for drug design. ► Occurrence of biotechnological interesting representatives in extremophiles.

Independent evolution of four heme peroxidase superfamilies

Graphical abstract

Evolution of structure and function of Class I peroxidases

The phylogenetics of Class I of the heme peroxidase-catalase superfamily currently representing over 940 known sequences in all available genomes of prokaryotes and eukaryotes has been analysed. The robust reconstructed tree for 193 Class I peroxidases with 6 selected Class II representatives reveals all main trends of molecular evolution. It suggests how the ancestral peroxidase gene might have been transferred from prokaryotic into eukaryotic genomes. Besides well known families of catalase-peroxidases, cytochrome c peroxidases and ascorbate peroxidases, the phylogenetic analysis shows for the first time the presence of two new well separated clades of hybrid-type peroxidases that might represent evolutionary bridges between catalase-peroxidases and cytochrome c peroxidases (type A) as well as between ascorbate peroxidases and Class II peroxidases (type B). Established structure-function relationships are summarized. Presented data give useful hints on the origin and evolution of catalytic promiscuity and specificity and will be a valuable basis for future functional analysis of Class I enzymes as well as for de novo design.

Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria)

Blue-green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant-type photosynthetic organisms. They were the first which produced molecular oxygen as a byproduct of photosynthetic activity. Also today they live in habitats with potentially damaging photooxidative conditions due to high irradiation and oxygen concentrations. Therefore, the cells must have evolved protective mechanisms to cope with reactive oxygen species produced by incomplete reduction of molecular oxygen via electron transport processes to prevent damage of biologically important macromolecules. Hydrogen peroxide and organic peroxides can be removed by enzymes called hydroperoxidases which on the one hand disproportionate it (catalases and catalase-peroxidases) and on the other hand use electron donors to reduce it to water or the corresponding alcohols. Until now the sequenced or partially sequenced genomes of six cyanobacteria are available in databases. Based on similarity searches and multiple sequence alignments, several cyanobacterial hydroperoxidases can be detected. All the cyanobacteria possess peroxiredoxins which use thioredoxin or other reduced thiols to get rid of hydrogen peroxide and lipid peroxides. Nearly all cyanobacteria contain an NADPH-dependent glutathione peroxidase-like protein which uses NADPH to reduce unsaturated fatty acid hydroperoxides. The best analyzed cyanobacterial antioxidative enzyme is the hemoprotein catalase-peroxidase which has a high catalase activity but concerning the sequence it is a typical peroxidase. Two species seem to encode a manganese-containing catalase and Nostoc punctiforme could use a monofunctional catalase. There are as well additional peroxidases encoded in cyanobacteria whose physiological relevance is unknown. 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Common phylogeny of catalase-peroxidases and ascorbate peroxidases

Catalase-peroxidases belong to Class I of the plant, fungal, bacterial peroxidase superfamily, together with yeast cytochrome c peroxidase and ascorbate peroxidases. Obviously these bifunctional enzymes arose via gene duplication of an ancestral hydroperoxidase. A 230-residues long homologous region exists in all eukaryotic members of Class I, which is present twice in both prokaryotic and archaeal catalase-peroxidases. The overall structure of eukaryotic Class I peroxidases may be retained in both halves of catalase-peroxidases, with major insertions in several loops, some of which may participate in inter-domain or inter-subunit interactions. Interspecies distances in unrooted phylogenetic trees, analysis of sequence similarities in distinct structural regions, as well as hydrophobic cluster analysis (HCA) suggest that one single tandem duplication had already occurred in the common ancestor prior to the segregation of the archaeal and eubacterial lines. The C-terminal halves of extant catalase-peroxidases clearly did not accumulate random changes, so prolonged periods of independent evolution of the duplicates can be ruled out. Fusion of both copies must have occurred still very early or even in the course of the duplication. We suggest that the sparse representatives of eukaryotic catalase-peroxidases go back to lateral gene transfer, and that, except for several fungi, only single copy hydroperoxidases occur in the eukaryotic lineage. The N-terminal halves of catalase-peroxidases, which reveal higher homology with the single-copy members of the superfamily, obviously are catalytically active, whereas the C-terminal halves of the bifunctional enzymes presumably control the access to the haem pocket and facilitate stable folding. The bifunctional nature of catalase-peroxidases can be ascribed to several unique sequence peculiarities conserved among all N-terminal halves, which most likely will affect the properties of both haem ligands.

Two distinct groups of fungal catalase/peroxidases

Catalase/peroxidases (KatGs) are bifunctional haem b-containing (Class I) peroxidases with overwhelming catalase activity and substantial peroxidase activity with various one-electron donors. These unique oxidoreductases evolved in ancestral bacteria revealing a complex gene-duplicated structure. Besides being found in numerous bacteria of all phyla, katG genes were also detected in genomes of lower eukaryotes, most prominently of sac and club fungi. Phylogenetic analysis demonstrates the occurrence of two distinct groups of fungal KatGs that differ in localization, structural and functional properties. Analysis of lateral gene transfer of bacterial katGs into fungal genomes reveals that the most probable progenitor was a katG from a bacteroidetes predecessor. The putative physiological role(s) of both fungal KatG groups is discussed with respect to known structure-function relationships in bacterial KatGs and is related with the acquisition of (phyto)pathogenicity in fungi.

Turning points in the evolution of peroxidase–catalase superfamily: molecular phylogeny of hybrid heme peroxidases

Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase–catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase–catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.

Eukaryotic extracellular catalase–peroxidase from Magnaporthe grisea – Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state & presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a Kd value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction.

High conformational stability of secreted eukaryotic catalase-peroxidases: answers from first crystal structure and unfolding studies.

Background: Eukaryotic secreted KatGs are bifunctional enzymes extensively found in phytopathogenic fungi. Results: Structural peculiarities, mainly in the N-terminal domain, dominate stability and other properties of eukaryotic secreted KatGs. Conclusion: The distinctive requirements of secreted KatGs depend on very specific and fully conserved structural features. Significance: This might be exploited to control plant fungal diseases that are jeopardizing food security worldwide. Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal +Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.