Fungal catalases: Function, phylogenetic origin and structure

Most fungi have several monofunctional heme-catalases. Filamentous ascomycetes (Pezizomycotina) have two types of large-size subunit catalases (L1 and L2). L2-type are usually induced by different stressors and are extracellular enzymes; those from the L1-type are not inducible and accumulate in asexual spores. L2 catalases are important for growth and the start of cell differentiation, while L1 are required for spore germination. In addition, pezizomycetes have one to four small-size subunit catalases. Yeasts (Saccharomycotina) do not have large-subunit catalases and generally have one peroxisomal and one cytosolic small-subunit catalase. Small-subunit catalases are inhibited by substrate while large-subunit catalases are activated by H(2)O(2). Some small-subunit catalases bind NADPH preventing inhibition by substrate. We present a phylogenetic analysis revealing one or two events of horizontal gene transfers from Actinobacteria to a fungal ancestor before fungal diversification, as the origin of large-size subunit catalases. Other possible horizontal transfers of small- and large-subunit catalases genes were detected and one from bacteria to the fungus Malassezia globosa was analyzed in detail. All L2-type catalases analyzed presented a secretion signal peptide. Mucorales preserved only L2-type catalases, with one containing a secretion signal if two or more are present. Basidiomycetes have only L1-type catalases, all lacking signal peptide. Fungal small-size catalases are related to animal catalases and probably evolved from a common ancestor. However, there are several groups of small-size catalases. In particular, a conserved group of fungal sequences resemble plant catalases, whose phylogenetic origin was traced to a group of bacteria. This group probably has the heme orientation of plant catalases and could in principle bind NADPH. From almost a hundred small-subunit catalases only one fourth has a peroxisomal localization signal and in fact many fungi lack a peroxisomal catalase. Catalases have a deep buried active site and H(2)O(2) has to go through a long passage to reach it. In all known structures of catalases, the major channel has common features, particularly in the straight and narrow final section that is positioned perpendicular to the heme. Besides, other conserved channels are present in catalases whose function remains to be elucidated. One of these channels intercommunicates the major channels from the two R-related subunits. In three of the four known large-subunits catalase structures, the heme b is partially transformed into heme d. In Neurospora crassa, this occurs in vivo and is related to oxidative stress conditions in which singlet oxygen is produced. A pure source of singlet oxygen oxidizes catalases purified from different sources and singlet oxygen quenchers prevent oxidation. A second modification is observed in N. crassa catalase-1, in which the tyrosine that forms the fifth coordination bound to the heme iron makes a covalent bond with a vicinal cysteine, similarly to the tyrosine-histidine bonding found in Escherichia coli hydroperoxidase II. Molecular dynamics has been used to determine how H(2)O(2) reaches the enzyme active site and how products exit the protein. We found that the bottleneck of the major channel seems to disappear in water and is wide open in the presence of substrate. Amino acid residues exhibiting an increased residence time for H(2)O(2) are abundant at the protein surface and at the entrances to the major channel. The net effect of this is an increased H(2)O(2)/H(2)O ratio in the major channel. Once in the final section of this channel, H(2)O(2) is retained and tends to occupy specific sites while water molecules have a higher turnover rate and occupy different sites. Despite the intense study of catalases our knowledge of this enzyme is still limited and in need of new studies and different approaches.     

Molecular evolution of maize catalases and their relationship to other eukaryotic and prokaryotic catalases

We have compared the nucleotide and protein sequences of the three maize catalase genes with other plant catalases to reconstruct the evolutionary relationship among these catalases. These sequences were also compared with other eukaryotic and prokaryotic catalases. Phylogenies based on distances and parsimony analysis show that all plant catalases derive from a common ancestral catalase gene and can be divided into three distinct groups. The first, and major, group includes maizeCatl, barleyCat1, riceCatB and most of the dicot catalases. The second group is an apparent dicot-specific catalase group encompassing the tobaccoCat2 and tomatoCat. The third is a monocot-specific catalase class including the maize Cat3, barley Cat2, and riceCatA. The maize Cat2 gene is loosely related to the first group. The distinctive features of monocot-specific catalases are their extreme high codon bias at the third position and low degree of sequence similarity to other plant catalases. Similarities in the intron positions for several plant catalase genes support the conclusion of derivation from a common ancestral gene. The similar intron position between bean catalases and human catalase implies that the animal and plant catalases might have derived from a common progenitor gene sequence. Correspondence to: J.G. Scandalios     

Phylogenetic relationships among prokaryotic and eukaryotic catalases

Seventy-four catalase protein sequences, including 29 bacterial, 8 fungal, 7 animal, and 30 plant sequences, were compiled, and 70 were used for phylogenetic reconstruction. The core of the resulting tree revealed unique, separate groups of plant and animal catalases, two groups of fungal catalases, and three groups of bacterial catalases. The only overlap of kingdoms occurred within one branch and involved fungal and bacterial large-subunit enzymes. The other fungal branch was closely linked to the group of animal enzymes. Group I bacterial catalases were more closely related to the plant enzymes and contained such diverse taxa as the Gram-positive Listeria seeligeri, Deinocococcus radiodurans, and gamma-proteobacteria. Group III bacterial sequences were more closely related to fungal and animal sequences and included enzymes from a broad range of bacteria including high- and low-GC Gram positives, proteobacteria, and a bacteroides species. Group II was composed of large-subunit catalases from diverse sources including Gram positives (low-GC Bacilli and high-GC Mycobacteria), proteobacteria, and species of the filamentous fungus Aspergillus. These data can be interpreted in terms of two gene duplication events that produced a minimum of three catalase gene family members that subsequently evolved in response to environmental demands. Horizontal gene transfer may have been responsible for the group II mixture of bacterial and fungal large-subunit catalases.      

Recent insights into microbial catalases: Isolation,production and purification

Catalase, an oxidoreductase enzyme, works as a detoxification system inside living cells against reactive oxygen species formed as a by-product of different metabolic reactions. The enzyme is found in a wide range of aerobic and anaerobic organisms. Catalase has also been employed in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to its other applications in textile, paper, food and pharmaceutical industries. New applications for catalases are constantly emerging thanks to their high turnover rate, distinct evolutionary origin, relatively simple and well-defined reaction mechanisms. The following review provides comprehensive information on isolation, production and purification of catalases with different techniques from various microbial sources along with their types, structure, mechanism of action and applications.     

Differential Accumulation of Salicylic Acid and Salicylic Acid-Sensitive Catalase in Different Rice Tissues

We previously proposed that salicylic acid (SA)-sensitive catalases serve as biological targets of SA in plant defense responses. To further examine the role of SA-sensitive catalases, we have analyzed the relationship between SA levels and SA sensitivity of catalases in different rice (Oryza sativa) tissues. We show here that, whereas rice shoots contain extremely high levels of free SA, as previously reported (I. Raskin, H. Skubatz, W. Tang, B.J.D. Meeuse [1990] Ann Bot 66: 369-373; P. Silverman, M. Seskar, D. Kanter, P. Schweizer, J.-P. Metraux, I. Raskin [1995] Plant Physiol 108: 633-639), rice roots and cell-suspension cultures have very low SA levels. Catalases from different rice tissues also exhibit differences in sensitivity to SA. Catalase from rice shoots is insensitive to SA, but roots and cell-suspension cultures contain SA-sensitive catalase. The difference in SA sensitivity of catalases from these different tissues correlates with the tissue-specific expression of two catalase genes, CatA and CatB, which encode highly distinctive catalase proteins. CatA, which encodes a catalase with relatively low sequence homology to the tobacco SA-sensitive catalases, is expressed at high levels exclusively in the shoots. On the other hand, in roots and cell-suspension cultures, with northern analysis we detected expression of only the CatB gene, which encodes a catalase with higher sequence homology to tobacco catalases. The role of catalases in mediating some of the SA-induced responses is discussed in light of these results and the recently defined mechanisms of catalase inhibition by SA.     

Roles of water in heme peroxidase and catalase mechanisms

A water molecule is coproduced with the Compound I intermediate in the reactions of native heme peroxidases and catalases with hydrogen peroxide. As a result of water release/rebinding from/to the coproduct formation site the Compound I intermediate may exist in two forms: a "wet" form, Compound I(H(2)O), in which a water molecule is present at or near the site of coproduct water formation, and Compound I, in which the coproduct water formation site is "dry." It is postulated that the absence or presence of a water molecule at this site provides the structural basis for a redox pathway switching mechanism, such that the transition states for 2-electron equivalent reduction of Compound I intermediates are accessible in the dry form, but that in the wet form only 1-electron equivalent processes are possible, unless release of water can be stimulated. This concept provides the basis of a general mechanism in which the classical functional distinction between catalases and peroxidases, as well as the more complex behavior observed in halide oxidation and halogenation reactions, appear as particular cases in which variations in the degree of retention of water at the coproduct formation site influence Compound I reactivity.     

The genetics of maintenance of an all-male lineage in the Squalius albuvnoides complex

Squalius alburnoides is a complex of minnows common to the Iberian Peninsula, consisting of two distinct forms. The most common form is comprised of diploid and polyploid asexual hybrids heterozygous for several diagnostic allozyme loci contributed by Squalius pyrenaicus or Squalius carolitertii and a missing ancestor. The other form is diploid and homozygous for the allele contributed by the missing ancestor at these same loci. Present results from analyses of sex ratio and cytochrome b sequences are not consistent with the evolutionary distinctiveness of this non-hybrid form and suggest that it represents an all-male lineage imbedded within an almost all-female complex. This all-male lineage allowed preservation of the ancestral paternal nuclear genome after the paternal ancestor became extinct in all or most drainages, withimportant evolutionary implications.     

Molecular identification, heterologous expression and properties of light-insensitive plant catalases

Most catalases are inactivated by light in a heme-sensitized and O2-dependent reaction. In leaves of the alpine plant Homogyne alpina and in the peroxisomal cores of Helianthus annuus, light-insensitive catalases were observed. For the catalases Hacat1 of H. alpina and HnncatA3 of H. annuus, cDNA clones were obtained. Expression of recombinant active enzymes in insect cells confirmed that they coded for light-insensitive catalases. Kinetic and catalytic properties of light-sensitive or light-insensitive catalases did not differ substantially. However, the specific activity of the latter was markedly lower. The light-insensitive catalase HaCAT-1 was not resistant against inactivation by superoxide. Amino acid sequences of the light-insensitive catalases HaCAT-1 and HNNCATA3 were highly identical. They showed only a few exceptional amino acid substitutions at positions that are highly conserved in other catalases. These appeared to be localized mainly in a surface cavity at the entrance of a minor channel leading to the central heme, suggesting that this region played some, though yet undefined, role for light sensitivity. While the replacement of a highly conserved His by Thr225 was the most unique substitution, a single exchange of His225 by Thr in the light-sensitive catalase SaCAT-1 by mutagenesis was not sufficient to reduce its sensitivity to photoinactivation.     

Isolation from Micrococcus sp. n. of a homogeneous heme-containing catalase and a crystalline protein with catalase activity

A method for isolation and purification of catalases from the culture of Micrococcus sp. n. grown under aeration conditions is described. Heme-containing catalase (I) and the protein possessing a catalase activity (II) were separated by fractionation with ammonium sulfate. The specific activity of the highly purified protein causing degradation of H2O2 is 200 times less than that of the heme-containing enzyme. The molecular weights of catalases I and II as determined by electrophoresis in polyacrylamide gel gradient 4/30% are 240000 and 130000, respectively. The method described is designed at rapid isolation of preparative amounts of catalases from Micrococcus sp. n.     

Structural analysis of NADPH depleted bovine liver catalase and its inhibitor complexes

To study the functional role of NADPH during mammalian catalase inhibition, the X-ray crystal structures of NADPH-depleted bovine liver catalase and its inhibitor complexes, cyanide and azide, determined at 2.8Å resolution. From the complex structures it is observed that subunits with and without an inhibitor/catalytic water molecule are linked by N-terminal domain swapping. Comparing mammalian- and fungal- catalases, we speculate that NADPH-depleted mammalian catalases may function as a domain-swapped dimer of dimers, especially during inactivation by inhibitors like cyanide and azide. We further speculate that in mammalian catalases the N-terminal hinge-loop region and α-helix is the structural element that senses NADPH binding. Although the above arguments are speculative and need further verification, as a whole our studies have opened up a new possibility, viz. that mammalian catalase acts as a domain-swapped dimer of dimers, especially during inhibitor binding. To generalize this concept to the formation of the inactive state in mammalian catalases in the absence of tightly bound NADPH molecules needs further exploration. The present study adds one more intriguing fact to the existing mysteries of mammalian catalases.     

Sequence of the Saccharomyces cerevisiae CTA1 gene and amino acid sequence of catalase A derived from it

The nucleotide sequence of a 2785-base-pair stretch of DNA containing the Saccharomyces cerevisiae catalase A (CTA1) gene has been determined. This gene contains an uninterrupted open reading frame encoding a protein of 515 amino acids (relative molecular mass 58,490). Catalase A, the peroxisomal catalase of S. cerevisiae was compared to the peroxisomal catalases from bovine liver and from Candida tropicalis and to the non-peroxisomal, presumably cytoplasmic, catalase T of S. cerevisiae. Whereas the peroxisomal catalases are almost colinear, three major insertions have to be introduced in the catalase T sequence to obtain an optimal fit with the other proteins. Catalase A is most closely related to the C. tropicalis enzyme. It is also more similar to the bovine liver catalase than to the second S. cerevisiae catalase. The differences between the two S. cerevisiae enzymes are most striking within four blocks of amino acids consisting of a total of 37 residues with high homology between the three peroxisomal, but low conservation between the S. cerevisiae catalases. The results obtained indicate that the peroxisomal catalases compared have very similar three-dimensional structures and might have similar targeting signals.     

Molecular and kinetic study of catalase-1, a durable large catalase of Neurospora crassa.

Catalase-1 (Cat-1), one of the two monofunctional catalases of Neurospora crassa, increases during asexual spore formation to constitute 0.6% of total protein in conidia. Cat-1 was purified 170-fold with a yield of 48% from conidiating cultures. Like most monofunctional catalases, Cat-1 is a homotetramer, resistant to inactivation by solvents, fully active over a pH range of 4-12, and inactivated by 3-amino-1,2,4-triazole. Unlike most monofunctional catalases, Cat-1 consists of 88 kDa monomers that are glycosylated with alpha-glucose and/or alpha-mannose, is unusually stable, and is not inactivated or inhibited by hydrogen peroxide. Cat-1 was more resistant than other catalases to heat inactivation and to high concentrations of salt and denaturants. Cat-1 exhibited unusual kinetics: at molar concentrations of hydrogen peroxide the apparent V was 10 times higher than at millimolar concentrations. Inactivation of Cat-1 activity with azide and hydroxylamine was according to first order kinetics, while cyanide at micromolar concentrations was a reversible competitive inhibitor.     

Non-heme manganese catalase--the 'other' catalase

Non-heme manganese catalases are widely distributed over microbial life and represent an environmentally important alternative to heme-containing catalases in antioxidant defense. Manganese catalases contain a binuclear manganese complex as their catalytic active site rather than a heme, and cycle between Mn(2)(II,II) and Mn(2)(III,III) states during turnover. X-ray crystallography has revealed the key structural elements of the binuclear manganese active site complex that can serve as the starting point for computational studies on the protein. Four manganese catalase enzymes have been isolated and characterized, and the enzyme appears to have a broad phylogenetic distribution including both bacteria and archae. More than 100 manganese catalase genes have been annotated in genomic databases, although the assignment of many of these putative manganese catalases needs to be experimentally verified. Iron limitation, exposure to low levels of peroxide stress, thermostability and cyanide resistance may provide the biological and environmental context for the occurrence of manganese catalases.     

Interaction between sodium n-dodecyl sulphate and chemically modified bovine catalases

A series of catalases have been prepared in which a proportion of the carboxyl groups of glutamate and aspartate residues have been amidated with glycinamide. The physical properties of the amidated catalases have been investigated with specific reference to their interaction with sodium n-dodecyl sulphate (SDS). Amidation leads to an increase in SDS binding at pH 6.4. Microcalorimetric measurements show that the exothermic enthalpy of interaction with SDS increases with the extent of amidation in acid solution (pH 3.2–6.4). The increase in exothermicity is compensated by a decrease in entropy since the average Gibbs energy of SDS binding is independent of the extent of amidation. At pH 3.2 where the catalase carboxyl groups are largely un-ionized amidation still increase the exothermicity of the interaction with SDS. It is suggested that at low pH the SDS anion interacts favourably with the resonance stabilized O-protonated form of amidated side chains.     

Human liver catalase: cloning,expression and characterization of monoclonal antibodies

We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.     

Structure of the Clade 1 catalase,CatF of Pseudomonas syringae,at 1.8 A resolution

Catalase CatF of Pseudomonas syringae has been identified phylogenetically as a clade 1 catalase, closely related to plant catalases, a group from which no structure has been determined. The structure of CatF has been refined at 1.8 A resolution by using X-ray synchrotron data collected from a crystal flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are, respectively, 18.3% and 24.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The crystallized enzyme is a homotetramer of subunits with 484 residues, some 26 residues shorter than predicted from the DNA sequence. Mass spectrometry analysis confirmed the absence of 26 N-terminal residues, possibly removed by a periplasmic transport system. The core structure of the CatF subunit was closely related to seven other catalases with root-mean-square deviations (RMSDs) of 368 core Calpha atoms of 0.99-1.30 A. The heme component of CatF is heme b in the same orientation that is found in Escherichia coli hydroperoxidase II, an orientation that is flipped 180 degrees with respect the orientation of the heme in bovine liver catalase. NADPH is not found in the structure of CatF because key residues required for nucleotide binding are missing; 2129 water molecules were refined into the model. Water occupancy in the main or perpendicular channel of CatF varied among the four subunits from two to five in the region between the heme and the conserved Asp150. A comparison of the water occupancy in this region with the same region in other catalases reveals significant differences among the catalases.     

Structure-Function Relationships in Fungal Large-Subunit Catalases

Neurospora crassa has two large-subunit catalases, CAT-1 and CAT-3. CAT-1 is associated with non-growing cells and accumulates particularly in asexual spores; CAT-3 is associated with growing cells and is induced under different stress conditions. It is our interest to elucidate the structure-function relationships in large-subunit catalases. Here we have determined the CAT-3 crystal structure and compared it with the previously determined CAT-1 structure. Similar to CAT-1, CAT-3 hydrogen peroxide (H₂O₂) saturation kinetics exhibited two components, consistent with the existence of two active sites: one saturated in the millimolar range and the other in the molar range. In the CAT-1 structure, we found three interesting features related to its unusual kinetics: (a) a constriction in the channel that conveys H₂O₂ to the active site; (b) a covalent bond between the tyrosine, which forms the fifth coordination bound to the iron of the heme, and a vicinal cysteine; (c) oxidation of the pyrrole ring III to form a cis-hydroxyl group in C5 and a cis-γ-spirolactone in C6. The site of heme oxidation marks the starts of the central channel that communicates to the central cavity and the shortest way products can exit the active site. CAT-3 has a similar constriction in its major channel, which could function as a gating system regulated by the H₂O₂ concentration before the gate. CAT-3 functional tyrosine is not covalently bonded, but has instead the electron relay mechanism described for the human catalase to divert electrons from it. Pyrrole ring III in CAT-3 is not oxidized as it is in other large-subunit catalases whose structure has been determined. Different in CAT-3 from these enzymes is an occupied central cavity. Results presented here indicate that CAT-3 and CAT-1 enzymes represent a functional group of catalases with distinctive structural characteristics that determine similar kinetics.     

The C-terminal domain of plant catalases. Implications for a glyoxysomal targeting sequence.

A castor bean (Ricinus communis cv. Hale) cDNA encoding catalase was cloned and sequenced. The cDNA encoding the carboxy-terminal domain of catalase was compared to the corresponding sequences of six other plant catalases. The deduced amino acid sequences were compared according to the chemical attributes of each amino acid within each carboxy-terminal domain. A tripeptide sequence having the chemical attributes of the peroxisomal targeting sequence [Gould, S.J., Keller, G.-A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell Biol. 108, 1657-1664] was common to all the glyoxysomal/peroxisomal plant catalases. This sequence motif was located six amino acids from the carboxy terminus of each of the plant catalases. An identical motif was also found within the carboxy-terminal domain of three mammalian catalases previously sequenced. We hypothesize that these motifs are at least part of the targeting mechanism for catalase entry into plant glyoxysomes/peroxisomes.     

Unusual Cys-Tyr covalent bond in a large catalase

Catalase-1, one of four catalase activities of Neurospora crassa, is associated with non-growing cells and accumulates in asexual spores. It is a large, tetrameric, highly efficient, and durable enzyme that is active even at molar concentrations of hydrogen peroxide. Catalase-1 is oxidized at the heme by singlet oxygen without significant effects on enzyme activity. Here we present the crystal structure of catalase-1 at 1.75A resolution. Compared to structures of other catalases of the large class, the main differences were found at the carboxy-terminal domain. The heme group is rotated 180 degrees around the alpha-gamma-meso carbon axis with respect to clade 3 small catalases. There is no co-ordination bond of the ferric ion at the heme distal side in catalase-1. The catalase-1 structure exhibited partial oxidation of heme b to heme d. Singlet oxygen, produced catalytically or by photosensitization, may hydroxylate C5 and C6 of pyrrole ring III with a subsequent formation of a gamma-spirolactone in C6. The modification site in catalases depends on the way dioxygen exits the protein: mainly through the central channel or the main channel in large and small catalases, respectively. The catalase-1 structure revealed an unusual covalent bond between a cysteine sulphur atom and the essential tyrosine residue of the proximal side of the active site. A peptide with the predicted theoretical mass of the two bound tryptic peptides was detected by mass spectrometry. A mechanism for the Cys-Tyr covalent bond formation is proposed. The tyrosine bound to the cysteine residue would be less prone to donate electrons to compound I to form compound II, explaining catalase-1 resistance to substrate inhibition and inactivation. An apparent constriction of the main channel at Ser198 lead us to propose a gate that opens the narrow part of the channel when there is sufficient hydrogen peroxide in the small cavity before the gate. This mechanism would explain the increase in catalytic velocity as the hydrogen peroxide concentration rises.