Complete amino acid sequence ofProteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site

The catalase ofProteus mirabilis PR, a peroxide-resistant (PR) mutant ofProteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-typeP. mirabilis catalase.     

Comparison of beef liver and Penicillium vitale catalases

The structures of Penicillium vitale and beef liver catalase have been determined to atomic resolution. Both catalases are tetrameric proteins with deeply buried heme groups. The amino acid sequence of beef liver catalase is known and contains (at least) 506 amino acid residues. Although the sequence of P. vitale catalase has not yet been determined chemically, 670 residues have been built into the 2 A resolution electron density map and have been given tentative assignments. A large portion of each catalase molecule (91% of residues in beef liver catalase and 68% of residues in P. vitale catalase) shows structural homology. The root-mean-square deviation between 458 equivalenced C alpha atoms is 1.17 A. The dissimilar parts include a small fragment of the N-terminal arm and an additional "flavodoxin-like" domain at the carboxy end of the polypeptide chain of P. vitale catalase. In contrast, beef liver catalase contains one bound NADP molecule per subunit in a position equivalent to the chain region, leading to the flavodoxin-like domain, of P. vitale catalase. The position and orientation of the buried heme group in the two catalases, relative to the mutually perpendicular molecular dyad axes, are identical within experimental error. A mostly hydrophobic channel leads to the buried heme group. The surface opening to the channel differs due to the different disposition of the amino-terminal arm and the presence of the additional flavodoxin-like domain in P. vitale catalase. Possible functional implications of these comparisons are discussed.     

Thermochemical studies on the interaction between sodium n-dodecyl sulphate and catalases

The enthalpies of interaction between bovine catalase and sodium n-dodecyl sulphate (SDS) in aqueous solutions of pH 3.2,6.4 and 10.0 have been measured over a range of SDS concentrations by microcalorimetry at 25°C. The enthalpies increase with decreasing pH and with increasing SDS concentration and largely arise from the interations between the anionic head group of SDS and the cactionic amino acid residues on the protein. Chemically modified catalase in which a proportion of carboxylic acid groups have been coupled with either glycine methyl ester or ethylenediamine have been prepared and characterized in terms of their enzymic activities, spectral properties and sedimentation behaviour. The enthalpies of interaction of these catalases with SDS have been studied at pH 6.4. The results of the experiments suggest that the enthalpies of interaction with SDS can be correlated with the ratio of cationic to anionic amino acid residues on the surface of the catalase molecules and hence the nominal net surface charge. The variation in the enthalpy of interaction of catalases with surface charge, as a consequence of variation in pH, differs from the variation with charge at constant pH possibly due to the thermal effect of proton binding to the catalase—complexes.     

Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution.

The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A. The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion. The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.     

Ultracentrifugation studies on the dissociation of chemically modified catalases

The dissociation of a series of bovine catalases, in which a proportion of the carboxylic acid groups of glutamic and aspartic acids have been chemically modified by coupling with glycine methyl ester (GME) or ethylenediamine (ED), has been investigated by sedimentation rate and equilibrium methods. Sedimentation equilibrium measurements on GME derivatives have been analysed in terms of a monomer-dimer-trimer- tetramer model. The results show that the association of monomeric (M₁) catalase subunits is consistent with the equilibria 4M₁?2M₂?M₄. The Gibbs energies of association at 284K of the monomeric subunit to dimes (M₂) and tetramers (M₄) were found to be in the range ? 28 to ? 30 kJ mol^?1 and ? 91 to ? 97 kJ mol ^?1, respectively. The Gibbs energy for association of dimer to tetramer is in the range ? 32 to ? 34 kJ mol^?1. Chemical modification of bovine catalase markedly increases its susceptibility to dissociation by sodium n-dodecyl sulphate (SDS) and sedimentation rate measurements suggest that the initial event on addition of SDS is the dissociation of the whole molecule to half-molecules     

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.     

Complete amino acid sequence ofProteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site

The catalase ofProteus mirabilis PR, a peroxide-resistant (PR) mutant ofProteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-typeP. mirabilis catalase.     

Comparative kinetic characterization of catalases of the genusPenicillum

Extracellular catalases produced by fungi of the genusPenicillium, i.e.,P. piceum, P. varians, andP. kapuscinskii, were purified by consecutive filtration of culture liquids. The maximum reaction rate of H₂O₂ decomposition, the Michaelis constants, and specific catalytic activities of isolated catalases were determined. The operational stability was characterized by the effective rate of catalase inactivation during enzymatic reaction (k _in at 30°C). The thermal stability was determined by the rate of enzyme thermal inactivation at 45°C (k _in ^* , s^-1). Catalase fromP. piceum displayed the maximum activity, which was higher than the activity of catalase from bovine liver. The operational stability of catalase fromP. piceum was twofold to threefold higher than the stability of catalase from bovine liver. The physicochemical characteristics of catalases of fungi are better than the characteristics of catalase from bovine liver and intracellular catalase of yeastC. boidinii.     

Effect of sodium dodecyl fulfate on the dissociation of bovine liver catalase.

Native bovine liver catalase [EC 1.11.1.6] and catalase acetylated with N-acetylimidazole (AI) both combined with sodium dodecyl sulfate (SDS) to form catalase-SDS complexes. The differences between native and acetylated catalase bound to SDS were investigated as regards enzymatic activity, absorption spectra, ORD and CD, sedimentation velocity and fluorescence spectra. It was found that the binding of SDS with both catalases depended on incubation time and SDS concentration, and that the acetylation of catalase had some protective effect on the denaturation of the molecule by SDS, which may be ascribed to a reduction of ionic interaction between SDS and the protein on acetylation. The native catalase was found to split into three smaller components on incubation with 1% SDS for 96 hr, whereas the acetylated catalase split into two smaller components. These smaller components were isolated by gel filtration through Sephadex G-100. The isolated components has estimated molecular weights of 60,000, 30,000, aide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.     

Production of Catalases by Aspergillus niger Isolates as a Response to Pollutant Stress by Heavy Metals

Isolates of Aspergillus niger, selected from the coal dust of a mine containing arsenic (As; 400 mg/kg) and from the river sediment of mine surroundings (As, 1651 mg/kg, Sb, 362 mg/kg), growing in minimal nitrate medium in the phase of hyphal development and spore formation, exhibited much higher levels of total catalase activity than the same species from the culture collection or a culture adapted to soil contaminated with As (5 mg/L). Electrophoretic resolution of catalases in cell-free extracts revealed three isozymes of catalases and production of individual isozymes was not significantly affected by stress environments. Exogenously added stressors (As⁵⁺, Cd²⁺, Cu²⁺) at final concentrations of 25 and 50 mg/L and H₂O₂ (20 or 40 mM) mostly stimulated production of catalases only in isolates from mines surroundings, and H₂O₂ and Hg²⁺ caused the disappearance of the smallest catalase I. Isolates exhibited a higher tolerance of the toxic effects of heavy metals and H₂O₂, as monitored by growth, than did the strain from the culture collection.     

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.     

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 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     

Binding site amino acid residues of jack fruit (artocarpus integrifolia) seed lectin: chemical modification and protein difference spectral studies

The effect of chemical modification of amino acid residues essential for sugar binding in the α-D-galactoside specific jack fruit (Artocarpus integrifolia) seed lectin and the protection of the residues by specific sugar from modification were studied. Citraconylation or maleylation of 75 % of its lysyl residues or acetylation of 70 % of the tyrosyl residues completely abolished sugar binding and agglutination without dissociation of subunits. 1-O-methyl α-D-galactoside could protect its essential lysyl and tyrosyl groups from modification. Tryptophan could not be detected in the protein. Difference absorption spectra on binding of the above sugar confirmed the role of tyrosine residues and showed an association constantK = 0.4 × 10³ M⁻¹. Data suggests that the lectin could be immobilized without any loss of sugar binding activity     

Cloning, characterization, and expression in Escherichia coli of a gene encoding Listeria seeligeri catalase, a bacterial enzyme highly homologous to mammalian catalases.

A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the gram-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DH5 alpha as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately 50 times that of the combined E. coli catalases. The nucleotide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of the L. seeligeri catalase gene.     

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.     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.     

On the nature and characteristics of the multiple forms of catalase in mouse liver.

In order to clarify the nature of the heterogeneity of mouse liver catalase, the enzyme was purified and characterized by several criteria. Absorption and sedimentation properties provided little indication of significant differences between the mouse liver enzyme and catalases from other mammalian sources which do not display multiplicity. A denotement of the nature of the variformity in mouse liver catalase was provided, however, by the demonstration that the heteromorphs may be interconverted under conditions which favor the addition or removal of sialic acid residues. It was also observed that CMP-sialic acid, together with microsomal extract, protected the supernatant (desialated) pool of catalase from inactivation upon storage; and that the pattern of multiplicity which was exhibited by the purified enzyme on isoelectric focusing, was considerably altered by incubation with neuraminidase. With regard to the individual characteristics of the separate forms of purified mouse liver catalase, significant differences were noticeable in relation to their isoelectric points, specific activities, heme content, and specific binding of [¹⁴C]aminotriazole.     

Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources

A clone harbouring the genomic DNA sequence for the peroxisomal catalase of an n-alkane-utilizable yeast, Candida tropicalis, has been isolated by the hybrid-selection method and confirmed with a probe of catalase partial cDNA. Nucleotide sequence analysis of the cloned DNA disclosed that the gene fragment coding for catalase had a length of 1455 base pairs (corresponding to 485 amino acids; m = 54937 Da), and that the size of this enzyme was the smallest among all catalases reported hitherto. No intervening sequence was found in this coding region and some portions coincided with the amino acid sequences obtained from the analysis of the purified catalase. The comparison with three peroxisomal catalases from rat liver, bovine liver and human kidney, and one cytosolic catalase from Saccharomyces cerevisiae has revealed that catalase from C. tropicalis was more homologous to the peroxisomal enzymes than to the cytosolic one. C. tropicalis used the codons of the high-expression type. Amino acid residues were all conserved at the active and heme-binding sites. In the N and C-terminal regions there was no characteristic signal sequence or consensus sequence. However, a noticeable region, which can be discriminated between peroxisomal and cytosolic catalases, was proposed.     

Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response

When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 μM H₂O₂, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent K _m of 40 mM and a V _max of 285,000 U (mg protein)^–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases. Received: 15 October 1997 / Accepted: 2 February 1998