The Molecular Chaperone Mechanism of the C-Terminal Domain of Large-Size Subunit Catalases.

Large-size subunit catalases (LSCs) have an additional C-terminal domain (CT) that is structurally similar to Hsp31 and DJ-1 proteins, which have molecular chaperone activity. The CT of LSCs derives from a bacterial Hsp31 protein. There are two CT dimers with inverted symmetry in LSCs, one dimer in each pole of the homotetrameric structure. We previously demonstrated the molecular chaperone activity of the CT of LSCs. Like other chaperones, LSCs are abundant proteins that are induced under stress conditions and during cell differentiation in bacteria and fungi. Here, we analyze the mechanism of the CT of LSCs as an unfolding enzyme. The dimeric form of catalase-3 (CAT-3) CT (TDC3) of Neurospora crassa presented the highest activity as compared to its monomeric form. A variant of the CAT-3 CT lacking the last 17 amino acid residues (TDC3Δ17aa), a loop containing hydrophobic and charged amino acid residues only, lost most of its unfolding activity. Substituting charged for hydrophobic residues or vice versa in this C-terminal loop diminished the molecular chaperone activity in all the mutant variants analyzed, indicating that these amino acid residues play a relevant role in its unfolding activity. These data suggest that the general unfolding mechanism of CAT-3 CT involves a dimer with an inverted symmetry, and hydrophobic and charged amino acid residues. Each tetramer has four sites of interaction with partially unfolded or misfolded proteins. LSCs preserve their catalase activity under different stress conditions and, at the same time, function as unfolding enzymes.

Monofunctional Heme-Catalases.

The review focuses on four issues that are critical for the understanding of monofunctional catalases. How hydrogen peroxide (H2O2) reaches the active site and outcompetes water molecules to be able to function at a very high rate is one of the issues examined. Part of the answer is a gate valve system that is instrumental to drive out solvent molecules from the final section of the main channel. A second issue relates to how the enzyme deals with an unproductive reactive compound I (Cpd I) intermediate. Peroxidatic two and one electron donors and the transfer of electrons to the active site from NADPH and other compounds are reviewed. The new ascribed catalase reactions are revised, indicating possible measurement pitfalls. A third issue concerns the heme b to heme d oxidation, why this reaction occurs only in some large-size subunit catalases (LSCs), and the possible role of singlet oxygen in this and other modifications. The formation of a covalent bond between the proximal tyrosine with the vicinal residue is analyzed. The last issue refers to the origin and function of the additional C-terminal domain (TD) of LSCs. The TD has a molecular chaperone activity that is traced to a gene fusion between a Hsp31-type chaperone and a small-size subunit catalase (SSC).

Large-Size Subunit Catalases Are Chimeric Proteins: A H2O2 Selecting Domain with Catalase Activity Fused to a Hsp31-Derived Domain Conferring Protein Stability and Chaperone Activity.

Bacterial and fungal large-size subunit catalases (LSCs) are like small-size subunit catalases (SSCs) but have an additional C-terminal domain (CT). The catalytic domain is conserved at both primary sequence and structural levels and its amino acid composition is optimized to select H2O2 over water. The CT is structurally conserved, has an amino acid composition similar to very stable proteins, confers high stability to LSCs, and has independent molecular chaperone activity. While heat and denaturing agents increased Neurospora crassa catalase-1 (CAT-1) activity, a CAT-1 version lacking the CT (C63) was no longer activated by these agents. The addition of catalase-3 (CAT-3) CT to the CAT-1 or CAT-3 catalase domains prevented their heat denaturation in vitro. Protein structural alignments indicated CT similarity with members of the DJ-1/PfpI superfamily and the CT dimers present in LSCs constitute a new type of symmetric dimer within this superfamily. However, only the bacterial Hsp31 proteins show sequence similarity to the bacterial and fungal catalase mobile coil (MC) and are phylogenetically related to MC_CT sequences. LSCs might have originated by fusion of SSC and Hsp31 encoding genes during early bacterial diversification, conferring at the same time great stability and molecular chaperone activity to the novel catalases.

A critical analysis on the conception of "Pre-existent gene expression programs" for cell differentiation and development.

A pre-existent gene expression program at the basis of cell differentiation and development is often assumed in the current scientific literature. Historically this conception is traced to the nineteen sixties of the last century, when various influential papers and scientific personalities imprinted their view drawing inspiration from informatics. The accepted model is that in the presence of certain external and/or internal signals, a cell initiates a pre-determined program of gene expression by which it becomes differentiated. Authors generally do not question the evidence for the existence of such a program. Here I review different aspects and consequences of this model to conclude that it is completely at odds with the literature of the last decades, which has given us a splendid view of the dynamics of the living cell as an auto-organizing complex unit that is far away from thermodynamical equilibrium. In this view there is no place for programs.

Chaperone activity of large-size subunit catalases.

Large-size subunit catalases (LSCs) have a C-terminal domain that is structurally similar to DJ-1 and Hsp31 proteins, which have well documented molecular chaperone activity. Like chaperones, LSCs are abundant proteins that are induced under stress conditions and during cell differentiation in different microorganisms. Here we document that the C-terminal domain of LSCs assist other proteins to preserve their active conformation. Heat, urea, or H2O2 denaturation of alcohol dehydrogenase was prevented by LSCs or the C-terminal domain of Catalase-3 (TDC3); in contrast, small-size subunit catalases (SSCs) or LSCs without the C-terminal domain (C3ΔTD or C63) did not have this effect. Similar results were obtained if the alcohol dehydrogenase was previously denatured by heat and then the different catalases or truncated enzymes were added. The TDC3 also protected both the C3ΔTD and the bovine liver catalase from heat denaturation. The chaperone activity of CAT-3 or the TDC3 increased survival of E. coli under different stress conditions whereas the C3ΔTD did not. It is concluded that the C-terminal domain of LSCs has a chaperone activity that is instrumental for cellular resistance to stress conditions, such as oxidative stress that leads to cell differentiation in filamentous fungi.

X-ray driven reduction of Cpd I of Catalase-3 from N. crassa reveals differential sensitivity of active sites and formation of ferrous state.

Catalases are biotechnologically relevant enzymes because of their applications in food technology, bioremediation, and biomedicine. The dismutation of hydrogen peroxide occurs in two steps; in the first one, the enzyme forms an oxidized compound I (Cpd I) and in the second one, the enzyme is reduced to the ferric state. In this research work, we analyzed the reduction of Cpd I by X-ray radiation damage during diffraction experiments in crystals of CAT-3, a Large-Size Subunit Catalase (LSC) from Neurospora crassa. A Multi-Crystal Data collection Strategy was applied in order to obtain the Cpd I structure at a resolution of 2.2 Å; this intermediate was highly sensitive to X-ray and was easily reduced at very low deposited radiation dose, causing breakage of the Fe=O bond. The comparison of the structures showed reduced intermediates and also evidenced the differential sensitivity per monomer. The resting ferric state was reduced to the ferrous state, an intermediate without a previous report in LSC. The chemically obtained Cpd I and the X-ray reduced intermediates were identified by UV-visible microspectrometry coupled to data collection. The differential sensitivity and the formation of a ferrous state are discussed, emphasizing the importance of the correct interpretation in the oxidation state of the iron heme.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established.

Structure, kinetics, molecular and redox properties of a cytosolic and developmentally regulated fungal catalase-peroxidase.

CAT-2, a cytosolic catalase-peroxidase (CP) from Neurospora crassa, which is induced during asexual spore formation, was heterologously expressed and characterized. CAT-2 had the Met-Tyr-Trp (M-Y-W) adduct required for catalase activity. Its KM for H2O2 was micromolar for peroxidase and millimolar for catalase activity. A Em = -158 mV reduction potential value was obtained and the Soret band shift suggested a mixture of low and high spin ferric iron. CAT-2 EPR spectrum at 10 K indicated an axial and a rhombic component. With peroxyacetic acid (PAA), formation of Compound I* was observed with EPR. CAT-2 homodimer crystallographic structure contained two K+ ions; Glu107 residues were displaced to bind them. CAT-2 showed the essential amino acid residues for activity in similar positions to other CPs. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. A perhydroxy modification of the indole nitrogen of Trp90 was oriented toward the catalytic His91. In contrast to cytochrome c peroxidase and ascorbate peroxidase, the catalase-peroxidase heme propionates are not exposed to the solvent. Together with other N. crassa enzymes that utilize H2O2 as a substrate, CAT-2 has many tryptophan and proline residues at its surface, probably related to H2O2 selection in water.

How catalase recognizes H2O2 in a sea of water.

Monofunctional heme-catalases have been studied for many decades but there is still an incomplete understanding of why such a large tetrameric protein with deeply buried active sites is required to accomplish such a simple reaction as H2 O2 dismutation. Catalase accomplishes this reaction at a high rate although water at 55 M is expected to compete with H2 O2 for the enzyme's active site. Using molecular dynamics simulations we addressed the question as to how catalase selects H2 O2 in water. Selection is accomplished through different mechanisms: higher residence time of H2 O2 in the vicinity of certain prevalent amino acid residues at the protein surface and substrate channel, coordinated motion of the main passage amino acids that is increased in the presence of H2 O2 , a gate valve mechanism consisting of the motion of two contiguous phenylalanine residues that drive water molecules out of the final section of the substrate channel, a hydrophobic barrier before the active site that was crossed more easily by H2 O2 which kept most of its hydrogen bonds while passing, and finally an increased residence time for H2 O2 at the active site. These mechanisms, based on the physicochemical differences between H2 O2 and water, provide an explanation as to why such a large tetrameric protein with deeply buried active sites is required to accomplish efficient H2 O2 dismutation.

Conformational stability and crystal packing: polymorphism in Neurospora crassa CAT-3.

Polymorphism is frequently observed from different crystallization conditions. In proteins, the effect on conformational variability is poorly documented, with only a few reported examples. Here, three polymorphic crystal structures determined for a large-subunit catalase, CAT-3 from Neurospora crassa, are reported. Two of them belonged to new space groups, P1 and P43212, and a third structure belonged to the same space group, P212121, as the previously deposited 2.3 Å resolution structure (PDB entry 3ej6), but had a higher resolution (1.95 Å). Comparisons between these polymorphic structures highlight the conformational stability of tetrameric CAT-3 and reveal a distortion in the tetrameric structure that has not previously been described.

A white collar 1-like protein mediates opposite regulatory functions in Mucor circinelloides.

Protein ubiquitylation plays a major role in the regulation of many cellular processes by altering the stability, localization or function of target proteins. CrgA is a protein of Mucor circinelloides that shows the characteristics of ubiquitin ligases and is involved in the regulation of carotenogenesis and asexual sporulation in this fungus. CrgA, which belongs to a poorly characterized group of proteins present in almost all eukaryotes, represses carotenogenesis through the proteolysis-independent mono- and di-ubiquitylation of Mcwc-1b, a White Collar-1-like protein which, when it is non-ubiquitylated, activates carotenogenesis. Using a proteomic approach, this work shows that the regulation of M. circinelloides vegetative development by CrgA is also mediated by Mcwc-1b, although, in this case, the non-ubiquitylated Mcwc-1b form acts as a repressor. High levels of a protein that contains a classical Rossmann-fold NAD(P)H/NAD(P)(+) binding domain for NAD(P)H binding and is similar to NmrA NADP(H) sensor-like proteins occur when Mcwc-1b is inactivated by ubiquitylation. A role for this protein in the regulation of sporulation is suggested because its over-expression suppresses the sporulation defect in a crgAΔ mutant. NmrA-like proteins are repressors that interact with GATA transcription factors and have been shown to be related to cell differentiation in Magnaporthe oryzae and Dictyostelium discoideum. This proteomic approach also revealed that CrgA regulates the carbon and energy metabolism and that Mcwc-1b is the main, but not the only, target of CrgA.

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.

Catalase evolved to concentrate H2O2 at its active site.

Catalase is a homo-tetrameric enzyme that has its heme active site deeply buried inside the protein. Its only substrate, hydrogen peroxide (H2O2), reaches the heme through a 45 A-long channel. Large-subunit catalases, but not small-subunit catalases, have a loop (gate loop) that interrupts the major channel. Two accesses lead to a gate that opens the final section of the channel to the heme; gates from the R-related subunits are interconnected. Using molecular dynamic simulations of the Neurospora crassa catalase-1 tetramer in a box of water (48,600 molecules) or 6M H2O2, it is shown that the number of H2O2 molecules augments at the surface of the protein and in the accesses to the gate and the final section of the channel. Increase in H2O2 is due to the prevalence and distribution of amino acids that have an increased residency for H2O2 (mainly histidine, proline and charged residues), which are localized at the protein surface and the accesses to the gate. In the section of the channel from the heme to the gate, turnover rate of water molecules was faster than for H2O2 and increased residence sites for water and H2O2 were determined. In the presence of H2O2, the exclusion of water molecules from a specific site suggests a mechanism that could contend with the competing activity of water, allowing for catalase high kinetic efficiency.

Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication.

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

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 (H(2)O(2)) 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 H(2)O(2) 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-gamma-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 H(2)O(2) 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.

NADPH oxidases NOX-1 and NOX-2 require the regulatory subunit NOR-1 to control cell differentiation and growth in Neurospora crassa.

We have proposed that reactive oxygen species (ROS) play essential roles in cell differentiation. Enzymes belonging to the NADPH oxidase (NOX) family produce superoxide in a regulated manner. We have identified three distinct NOX subfamilies in the fungal kingdom and have shown that NoxA is required for sexual cell differentiation in Aspergillus nidulans. Here we show that Neurospora crassa NOX-1 elimination results in complete female sterility, decreased asexual development, and reduction of hyphal growth. The lack of NOX-2 did not affect any of these processes but led instead to the production of sexual spores that failed to germinate, even in the presence of exogenous oxidants. The elimination of NOR-1, an ortholog of the mammalian Nox2 regulatory subunit gp67(phox), also caused female sterility, the production of unviable sexual spores, and a decrease in asexual development and hyphal growth. These results indicate that NOR-1 is required for NOX-1 and NOX-2 functions at different developmental stages and establish a link between NOX-generated ROS and the regulation of growth. Indeed, NOX-1 was required for the increased asexual sporulation previously observed in mutants without catalase CAT-3. We also analyzed the function of the penta-EF calcium-binding domain protein PEF-1 in N. crassa. Deletion of pef-1 resulted in increased conidiation but, in contrast to what occurs in Dictyostelium discoideum, the mutation of this peflin did not suppress the phenotypes caused by the lack of NOX-1. Our results support the role of ROS as critical cell differentiation signals and highlight a novel role for ROS in regulation of fungal growth.

Fungal responses to reactive oxygen species.

Reactive oxygen species (ROS) such as hydrogen peroxide, produced externally or during normal metabolism, can damage different cell components and usually trigger a counteracting antioxidant response. The fact that animals and humans utilize ROS and related nitrogen reactive species to prevent fungal infection has generated great interest in defining the components of the antioxidant response and studying their role as virulence determinants in fungi. Here we review the role of specific enzyme and non-enzyme mediated antioxidant mechanisms in virulence, as well as the signal transduction mechanisms that fungal cells use to perceive high ROS levels and induce gene expression. We focus on Schizosaccharomyces pombe antioxidant responses, which involve a prokaryotic-type multistep phosphorelay coupled to a stress-response MAP kinase pathway and an AP-1 type transcription factor, in relation to homologous mechanisms in Aspergillus nidulans and the human pathogen A. fumigatus. Compared to S. pombe and other unicellular fungi, filamentous fungi have additional mechanisms to handle ROS, such as the presence of a larger number of phosphorelay sensor kinases, antioxidant enzymes and secondary metabolites with antioxidant functions. In addition, filamentous fungi have enzymes like the NADPH oxidases, which regulate multicellular development through ROS production and therefore, offer a unique opportunity to study the interplay between ROS production, perception and detoxification, and the role of these processes in cell differentiation and pathogenesis.

Functional and structural analysis of catalase oxidized by singlet oxygen.

Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H(2)O(2) concentration range (0.01-3.0 M), non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to high temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is structurally different from the non-oxidized one, although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase could have been selected through evolution to assure an active enzyme under oxidative stress conditions.

Reactive oxygen species and development in microbial eukaryotes.

Reactive oxygen species (ROS) have been regarded as inevitable harmful by-products of aerobic metabolism. Growing evidence, however, suggests that ROS play important physiological roles. This raises questions about the pathways that different groups of organisms use to produce and sense ROS. In microbial eukaryotes, recent data show (i) increased ROS levels during cell differentiation, (ii) the existence of ROS-producing enzymes, such as NADPH oxidases (NOX), (iii) the involvement of NOX in developmental processes, and (iv) a conservation in the signal-transduction mechanisms used to detect ROS. This shows that manipulation of reactive species, as strategy to regulate cell differentiation, is ubiquitous in eukaryotes and suggests that such strategy was selected early in evolution.

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.