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.     

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.     

玉米FAD2基因的克隆及序列分析

高等植物中的A12脂肪酸脱饱和酶是将油酸转化为亚油酸的酶。根据已发表的其他高等植物的FAD2基因的保守序列设计同源引物,通过RT—PCR从玉米幼胚中扩增得到一个特异的cDNA基因片段。通过生物信息学分析,从玉米幼胚cDNA和基因组中均扩增得到1164 bp FAD2基因（GenBank登陆号：DQ496227）,它编码387个氨基酸,含有完整的ORF框,在ORF框内无内含子。序列联配与树状分析结果表明,FAD2推导的氨基酸序列与其他物种的A12脱饱和酶基因具有同源性。它含有3个组氨酸保守域和2段很长的疏水区,是一个跨膜4次的膜结合蛋白。半定量RT—PCR分析显示FAD2基因在玉米幼胚中表达量最高,在叶、茎、根中亦有低水平表达。     

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.     

Purification,characterization, and gene sequencing of a catalase from an alkali- and halo-tolerant bacterium,Halomonas sp. SK1

An alkali- and halo-tolerant bacterium with high catalase activity was isolated and identified as a new species of the genus Halomonas. Its catalase (HktA) was simply purified by two steps of liquid chromatography. A 71.4% yield of the catalase was obtained with 97% purity on SDS-PAGE. The specific activity of HktA (57,900 U/mg protein) was two times higher than that of bovine liver catalase. The purified enzyme is inhibited by KCN, NH2OH, NaN3, and 3-amino-1,2,4-triazole, active at pH 5.0-11.0, thermo-sensitive, and KCl-tolerant. HktA is suggested to be a typical catalase, a homotetrameric protein containing heme groups in the active sites. The nucleotide sequence of the catalase gene (hktA) comprises 1,530 bp, encoding a protein of 509 amino acid residues. The deduced amino acid sequence of the hktA shares 99% identity with that of Vibrio rumoiensis S-1T.     

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     

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146bp with a 5' UTR of 103bp, an unusually long 3' UTR of 1519bp with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 1521bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12h post-Vibrio infection, then recovered to the original level at 24h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop.     

甘蔗过氧化氢酶基因的电子克隆及生物信息学分析

应用电子克隆技术,获得甘蔗中一个过氧化氢酶基因的cDNA序列全长,命名为S-CAT。该基因全长2160bp,包含一个1479bp的开放阅读框,编码492个氨基酸。通过PSORT工具,预测甘蔗S-CAT蛋白存在于过氧化物酶体中。同源比对分析显示,S-CAT基因编码的氨基酸序列与水稻、玉米、高粱、朝鲜碱茅、葡萄等植物中过氧化氢酶基因所编码的氨基酸序列高度同源,同源性分别为97%,97%,95%,91%和92%。研究结果为该基因的实验克隆奠定基础。     

Molecular cloning,characterization and expression analysis of two catalase isozyme genes in barley

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).     

The profilin multigene family of maize: differential expression of three isoforms

Profilin is a small (12–15 kDa) actin- and phospholipid-binding protein previously known only from studies on animals and lower eukaryotes but recently identified as a birch pollen allergen. Here we have identified and characterized three members of the profilin multigene family from the plant Zea mays . Two cDNAs isolated from a maize pollen library ( ZmPRO 1 and ZmPRO 3) each have a single, large open reading frame encoding a putative polypeptide 131 amino acids long with a predicted molecular weight of approximately 14 kDa. A third maize pollen cDNA ( ZmPRO 2) has two in-frame translation initiation codons. Use of the first ATG would result in a polypeptide 137 amino acids long with a molecular weight of 14.8 kDa. The three maize profilins are highly homologous to each other (>90% nucleotide and amino acid sequence identity) as well as other plant profilins but show far less similarity (30–40% amino acid sequence identity) to animal and lower eukaryote profilins. Multiple sequence alignments indicate that only nine residues are shared by all eukaryotic profilins examined. However, limited comparisons reveal domains in the NH₂ and COOH termini that have a high degree of similarity suggesting functional conservation. The maize gene family size is estimated to contain three to six members based on Southern blot experiments with gene-specific and coding region probes. Northern blot analysis demonstrates that the three maize profilin cDNAs characterized here are utilized in a tissue-specific manner and are anther or pollen specific.     

Two genes encode the two subunits of cottonseed catalase

The isolation and sequence of a cDNA encoding a developmentally distinct subunit of cottonseed catalase are presented. A 1.8-kb cDNA was selected from a cDNA library constructed with poly(A)+ RNA isolated from 3-day-old dark-grown cotyledons in which a second subunit (designated SU 2 in an earlier publication) of catalase was predominantly synthesized. The cDNA encodes a 492-amino acid peptide with a calculated Mr of 56,900. The nucleotide sequence is 76% identical to a cDNA encoding another subunit (SU 1) which was predominantly synthesized in 1-day-old-cotyledons. Most of the divergence occurs in the 5' and 3' non-coding regions, and at the third positions of the codons. The deduced amino acid sequence is 92% identical to that of SU 1. Denaturing isoelectric focusing and SDS-PAGE of products transcribed and translated in vitro from these cDNAs revealed that the cDNA selected from the "1-day" library encoded SU 1 and the cDNA selected from the "3-day" library (this paper) encoded SU 2 of catalase. These data and results from Southern blot analyses of genomic DNA indicate that there are two genes encoding catalase subunits in cotton cotyledons, with only one copy of SU 1 and at least two copies of SU 2 in the genome. A peroxisomal targeting signal, e.g., Ser-Lys-Leu, is not located at the C-terminus of either subunit, or within 25 residues of the C-terminus of SU 1, although it occurs at six residues upstream from the C-terminus of SU 2. A possible location of a targeting sequence for catalase and other peroxisomal proteins lacking the C-terminal tripeptide motif is proposed.     

Relationship between the size of the bottleneck 15 A from iron in the main channel and the reactivity of catalase corresponding to the molecular size of substrates

A catalase that exhibits a high level of activity and a rapid reaction with organic peroxides has been purified from Exiguobacterium oxidotolerans T-2-2T (EKTA catalase). The amino acid sequence of EKTA catalase revealed that it is a novel clade 1 catalase. Amino acid residues in the active site around the protoheme are conserved in the primary structure of EKTA catalase. Although the general interactions of molecules larger than hydrogen peroxide with catalases are strongly inhibited because of the selection role of long and narrow channels in the substrate reaching the active site, the formation rate of reactive intermediates (compound I) in the reaction of EKTA catalase with peracetic acid is 77 times higher than that of bovine liver catalase (BLC) and 1200 times higher than that of Micrococcus luteus catalase (MLC). The crystal structure of EKTA catalase has been determined and refined to 2.4 A resolution. The main channel structure of EKTA catalase is different from those of BLC and MLC. The rate constant of compound I formation in catalases decreased with an increase in the molecular size of the substrate. For EKTA catalase with a larger bottleneck 15 A from the iron (entrance of narrow channel) in the main channel, a lower rate of reduction in compound I formation rate with an increase in the molecular size of substrates was found. The increase in the rate constant of compound I formation in these catalases was directly proportional to the increase in the size of the bottleneck in the main channel when molecules of substrates larger than H2O2, such as organic peroxides, are used in the reaction. The results indicate that the size of the bottleneck in the main channel in catalase is an important factor in defining the rate of compound I formation corresponding to the molecular size of the substrates, and this was demonstrated. The Leu149-Ile180 and Asp109-Met167 combinations at the entrance of the narrow channel in EKTA catalase determine the size of the bottleneck, and each atom-to-atom distance for the combination of residues was larger than those of corresponding combinations of amino acid residues in BLC and MLC. The combination of these four amino acids is quite specific in EKTA catalase as compared with the combinations in other catalases in the gene database (compared with more than 432 catalase genes in the database).     

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.     

Cloning and sequencing of a cDNA encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase from maize. Possible relationship to the alkaline phosphatase family.

The primary sequence of maize 2,3-bisphosphoglycerate-independent phosphoglycerate mutase was deduced from cDNAs isolated from maize cDNA libraries by screening with specific antibodies to the cofactor-independent enzyme and from a maize genomic clone. The genomic clone provided the 5'-nucleotide sequence encoding the N-terminal amino acids which could not be obtained from the cDNA. Confirmation that the nucleotide sequence was for the cofactor-independent phosphoglycerate mutase was obtained by sequencing the peptides generated from cyanogen bromide cleavage of the purified protein. This is the first report of the amino acid sequence of a 2,3-bisphosphoglycerate cofactor-independent phosphoglycerate mutase, which consists of 559 amino acids and is twice the molecular size of the mammalian cofactor-dependent enzyme subunit. Analysis of the cofactor-independent phosphoglycerate mutase amino acid sequence revealed no identity with the cofactor-dependent mutase types. Northern blot analysis confirmed this difference since the maize cofactor-independent phosphoglycerate mutase cDNA did not hybridize with mRNA of the cofactor-dependent mutase. The lack of amino acid identity between cofactor-dependent and -independent enzymes is consistent with their different catalytic mechanisms and suggests that both enzymes are unrelated evolutionarily and arose from two independent ancestral genes. However, a constellation of residues which are involved in metal ion binding in various alkaline phosphatases is conserved in the maize cofactor-independent phosphoglycerate mutase, which suggests that the enzyme is a member of the alkaline phosphatase family of enzymes.     

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.     

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.     

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.     

从人血液中克隆过氧化氢酶基因

目的从人血液中克隆过氧化氢酶基因。方法以新鲜的人血液为材料,提取全血RNA,运用RT—PCR方法扩增过氧化氢酶基因,构建pMDl8T／CAT克隆载体,并进行了不同来源过氧化氢酶的氨基酸序列同源分析。结果从人血液中成功扩增出过氧化氢酶基因,获得了重组载体PMD-18T／CAT,氨基酸序列分析的同源性达80％以上。结论从人血液中可以很方便地克隆出过氧化氢酶基因。     

IDENTIFICATION AND CHARACTERIZATION OF THE CATALASE GENE PyCAT FROM THE RED ALGA PYROPIA YEZOENSIS (BANGIALES,RHODOPHYTA)1

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by reducing hydrogen peroxide. The full‐length catalase cDNA sequence as isolated from expressed sequence tags (ESTs) of Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (PyCAT) through rapid amplification of cDNA ends (RACE) was identified and characterized. It encoded a polypeptide of 529 amino acids, which shared 36%–44% similarity with other known catalase proteins. Phylogenetic analysis revealed that PyCAT was closer to the catalases from plants than from other organisms. The PyCAT mRNA expression was investigated using real‐time PCR to determine life‐cycle‐specific expression and the expression pattern during desiccation. The mRNA expression level in gametophytes was significantly higher than in sporophytes, and the mRNA expression level of PyCAT was significantly up‐regulated during the desiccation process. The recombinant PyCAT protein was purified and analyzed biochemically. The recombinant PyCAT protein exhibited high enzymatic activity (28,000 U·mg^?1) with high thermal stability and a broad pH range. All these results indicate that the PyCAT is a typical member of the plant and algal catalase family and may play a significant role in minimizing the effect of oxidative damage in P. yezoensis during desiccation.