Understanding the structure and function of catalases: clues from molecular evolution and in vitro mutagenesis.

This review gives an overview about the structural organisation of different evolutionary lines of all enzymes capable of efficient dismutation of hydrogen peroxide. Major potential applications in biotechnology and clinical medicine justify further investigations. According to structural and functional similarities catalases can be divided in three subgroups. Typical catalases are homotetrameric haem proteins. The three-dimensional structure of six representatives has been resolved to atomic resolution. The central core of each subunit reveals a characteristic "catalase fold", extremely well conserved among this group. In the native tetramer structure pairs of subunits tightly interact via exchange of their N-terminal arms. This pseudo-knot structures implies a highly ordered assembly pathway. A minor subgroup ("large catalases") possesses an extra flavodoxin-like C-terminal domain. A > or = 25 A long channel leads from the enzyme surface to the deeply buried active site. It enables rapid and selective diffusion of the substrates to the active center. In several catalases NADPH is tightly bound close to the surface. This cofactor may prevent and reverse the formation of compound II, an inactive reaction intermediate. Bifunctional catalase-peroxidase are haem proteins which probably arose via gene duplication of an ancestral peroxidase gene. No detailed structural information is currently available. Even less is know about manganese catalases. Their di-manganese reaction centers may be evolutionary.     

Mammalian genome evolution: new clues from comparisons of eutherians, marsupials and monotremes.

1. Comparisons of chromosomes and gene maps of different mammals are yielding a big picture of the evolution of mammalian genome form and function. It has been particularly instructive to compare gene arrangements on the sex chromosomes between the three major groups of mammals. Eutheria (so-called placental mammals). Metatheria (marsupials) and Prototheria (monotremes), which diverged 150 and 170 Myr BP respectively. 2. A region amounting to 3% of the haploid genome is located on the X chromosome in all three groups, implying that this region must have been part of the original X in a common ancestor. This region comprises the long arm of the human X. 3. A region represented by the short arm of the human X is common to the X in all eutherians, but is autosomal in marsupials and monotremes; thus it was not a part of the original X, and must have been acquired by the X early in the eutherian radiation. 4. This recently acquired region was probably translocated to a pseudoautosomal region shared by the eutherian X and Y. Thus it was originally paired and exempt from X chromosome inactivation; stepwise deletion of this region from the Y and recruitment of the newly unpaired region of the X into the inactivation system could account for some of the peculiarities of this region of the human X. 5. The sex-determining gene TDF must lie on the Y in all mammals in which the Y is male determining. The autosomal location of the candidate gene ZFY in marsupials and monotremes eliminates it from consideration. The recently described candidate gene SRY has yet to pass the "marsupial test".     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5′CYCGRG3′ and cleave between the first C and second Y to generate a four-base 5′ extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the “endo-blue” method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

Understanding the association of apolipoprotein E4 with Alzheimer disease: clues from its structure

Despite intense interest, the molecular mechanisms underlying the association of apoE4 with Alzheimer disease are not clear. Because the function (or dysfunction) of a protein is based on its structure, this review focuses on the effects of the structural differences among the isoforms on neurodegeneration. Understanding how apoE4 structure impacts neurodegeneration is likely to provide mechanistic insight as well as potential therapeutic approaches to blunt or reduce its effects.     

The human olfactory subgenome: from sequence to structure and evolution

Olfactory receptors (ORs) constitute the largest multigene family in multicellular organisms. Their evolutionary proliferation has been driven by the need to provide recognition capacity for millions of potential odorants with arbitrary chemical configurations. Human genome sequencing has provided a highly informative picture of the "olfactory subgenome", the repertoire of OR genes. We describe here an analysis of 224 human OR genes, a much larger number than hitherto systematically analyzed. These are derived by literature survey, data mining at 14 genomic clusters, and by an OR-targeted experimental sequencing strategy. The presented set contains at least 53% pseudogenes and is minimally divided into 11 gene families. One of these (no. 7) has undergone a particularly extensive expansion in primates. The analysis of this collection leads to insight into the origin of OR genes, suggesting a graded expansion through mammalian evolution. It also allows us to delineate a structural map of the respective proteins. A sequence database and analysis package is provided (http://bioinformatics.weizmann.ac.il/HORDE), which will be useful for analyzing human OR sequences genome-wide.     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5CYCGRG3 and cleave between the first C and second Y to generate a four-base 5 extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the endo-blue method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.     

Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems

Restriction modification (RM) systems serve to protect bacteria against bacteriophages. They comprise a restriction endonuclease activity that specifically cleaves DNA and a corresponding methyltransferase activity that specifically methylates the DNA, thereby protecting it from cleavage. Such systems are very common in bacteria. To find out whether the widespread distribution of RM systems is due to horizontal gene transfer, we have compared the codon usages of 29 type II RM systems with the average codon usage of their respective bacterial hosts. Pronounced deviations in codon usage were found in six cases:EcoRI,EcoRV,KpnI,SinI,SmaI, andTthHB81. They are interpreted as evidence for horizontal gene transfer in these cases. As the methodology is expected to detect only one-fourth to one-third of all horizontal gene transfer events, this result implies that horizontal gene transfer had a considerable influence on the distribution and evolution of RM systems. In all of these six cases the codon usage deviations of the restriction enzyme genes are much more pronounced than those of the methyltransferase genes. This result suggests that in these cases horizontal gene transfer had occurred sequentially with the gene for the methyltransferase being first acquired by the cell. This can be explained by the fact that an active restriction endonuclease is highly toxic in cells whose DNA is not protected from cleavage by a corresponding methyltransferase.     

Trends in protein evolution inferred from sequence and structure analysis

Complementary developments in comparative genomics, protein structure determination and in-depth comparison of protein sequences and structures have provided a better understanding of the prevailing trends in the emergence and diversification of protein domains. The investigation of deep relationships among different classes of proteins involved in key cellular functions, such as nucleic acid polymerases and other nucleotide-dependent enzymes, indicates that a substantial set of diverse protein domains evolved within the primordial, ribozyme-dominated RNA world.     

MARNA: multiple alignment and consensus structure prediction of RNAs based on sequence structure comparisons

MOTIVATION: Due to the importance of considering secondary structures in aligning functional RNAs, several pairwise sequence-structure alignment methods have been developed. They use extended alignment scores that evaluate secondary structure information in addition to sequence information. However, two problems for the multiple alignment step remain. First, how to combine pairwise sequence-structure alignments into a multiple alignment and second, how to generate secondary structure information for sequences whose explicit structural information is missing. RESULTS: We describe a novel approach for multiple alignment of RNAs (MARNA) taking into consideration both the primary and the secondary structures. It is based on pairwise sequence-structure comparisons of RNAs. From these sequence-structure alignments, libraries of weighted alignment edges are generated. The weights reflect the sequential and structural conservation. For sequences whose secondary structures are missing, the libraries are generated by sampling low energy conformations. The libraries are then processed by the T-Coffee system, which is a consistency based multiple alignment method. Furthermore, we are able to extract a consensus-sequence and -structure from a multiple alignment. We have successfully tested MARNA on several datasets taken from the Rfam database.     

Cloning, primary structure determination and comparative analysis of DNA-methyltransferases from SfaNI and Bst19I restriction-modification systems

Genes encoding DNA-methyltransferases which recognize the same sequence 5'-GCATC-3' from SfaNI and Bst19I restriction-modification systems have been cloned and primary structures of these have been determined. It has been revealed that restriction-modification system Bst19I contains two DNA-methyltransferases M1.Bst19I and M2.Bst19I, whereas RM system SfaNI include only one DNA-methyltransferase M.SfaNI, N- and C-domain of which are homologous of M2.Bst19I and M1.Bst19I, respectively. M1.Bst19I and M2.Bst19I as well as both domains of M.SfaNI contain conservative elements in an order that is typical for N6-adenine DNA-methyltransferases alpha class. SfaNI and Bst19I DNA-methyltransferases share high homology level with methylases of FokI and BstF5I RM systems. Probably this reflects presence of the common DNA sequence 5'-GATG-3' in the recognition sites of all these RM systems. Basing on primary structures homology of methylases, highly conserved amino acid residues on known spatial model of DNA-methyltransferase M.DpnIIA have been determined.     

Cloning of the HhaI and HinPI restriction-modification systems

The genes for the HhaI (Roberts et al., 1976) and HinPI (Roberts, 1987) restriction-modification (R-M) systems have been cloned in pBR322. The HhaI system was isolated on a 9-kb PstI fragment, and the HinPI system was isolated on two PstI fragments of 1.5 and 4.6 kb in length. The clones were isolated by selecting for recombinant molecules that had protectively modified themselves. The HhaI and HinPI R-M systems recognize the same sequence, GCGC, but hybridization between the DNA fragments encoding them does not take place.     

Organization of restriction-modification systems.

The genes for over 100 restriction-modification systems have now been cloned, and approximately one-half have been sequenced. Despite their similar function, they are exceedingly heterogeneous. The heterogeneity is evident at three levels: in the gene arrangements; in the enzyme compositions; and in the protein sequences. This paper summarizes the main features of the R-M systems that have been cloned.     

Structure and evolution of the XcyI restriction-modification system.

The XcyI restriction-modification system from Xanthomonas cyanopsidis recognizes the sequence, CCCGGG. The XcyI endonuclease and methylase genes have been cloned and sequenced and were found to be aligned in a head to tail orientation with the methylase preceding and overlapping the endonuclease by one base pair. The nucleotide sequence codes for an N4 cytosine methyltransferase with a predicted molecular weight of 33,500 and an endonuclease comprised of 333 codons and a molecular weight of 36,600. Sequence comparisons revealed significant similarity between the XcyI, CfrI and SmaI methylisomers. In contrast, no similarity was detected between the primary structures of the XcyI and SmaI endonucleases. The XcyI restriction-modification system is highly homologous to the XmaI genes, although the DNA sequences flanking the genes rapidly diverge. The sequence of the XcyI endonuclease contains two motifs which have recently been identified as essential to the activity of the EcoRV endonuclease.     

Stability of EcoRI restriction-modification enzymes in vivo differentiates the EcoRI restriction-modification system from other postsegregational cell killing systems

Certain type II restriction modification gene systems can kill host cells when these gene systems are eliminated from the host cells. Such ability to cause postsegregational killing of host cells is the feature of bacterial addiction modules, each of which consists of toxin and antitoxin genes. With these addiction modules, the differential stability of toxin and antitoxin molecules in cells plays an essential role in the execution of postsegregational killing. We here examined in vivo stability of the EcoRI restriction enzyme (toxin) and modification enzyme (antitoxin), the gene system of which has previously been shown to cause postsegregational host killing in Escherichia coli. Using two different methods, namely, quantitative Western blot analysis and pulse-chase immunoprecipitation analysis, we demonstrated that both the EcoRI restriction enzyme and modification enzyme are as stable as bulk cellular proteins and that there is no marked difference in their stability. The numbers of EcoRI restriction and modification enzyme molecules present in a host cell during the steady-state growth were estimated. We monitored changes in cellular levels of the EcoRI restriction and modification enzymes during the postsegregational killing. Results from these analyses together suggest that the EcoRI gene system does not rely on differential stability between the toxin and the antitoxin molecules for execution of postsegregational cell killing. Our results provide insights into the mechanism of postsegregational killing by restriction-modification systems, which seems to be distinct from mechanisms of postsegregational killing by other bacterial addiction modules.     

The immunoglobulin fold family: sequence analysis and 3D structure comparisons.

Fifty-two 3D structures of Ig-like domains covering the immunoglobulin fold family (IgFF) were compared and classified according to the conservation of their secondary structures. Members of the IgFF are distantly related proteins or evolutionarily unrelated proteins with a similar fold, the Ig fold. In this paper, a multiple structural alignment of the conserved common core is described and the correlation between corresponding sequences is discussed. While the members of the IgFF exhibit wide heterogeneity in terms of tissue and species distribution or functional implications, the 3D structures of these domains are far more conserved than their sequences. We define topologically equivalent residues in the Ig-like domains, describe the hydrophobic common cores and discuss the presence of additional strands. The disulfide bridges, not necessary for the stability of the Ig fold, may have an effect on the compactness of the domains. Based upon sequence and structure analysis, we propose the introduction of two new subtypes (C3 and C4) to the previous classifications, in addition to a new global structural classification. The very low mean sequence identity between subgroups of the IgFF suggests the occurrence of both divergent and convergent evolutionary processes, explaining the wide diversity of the superfamily. Finally, this review suggest that hydrophobic residues constituting the common hydrophobic cores are important clues to explain how highly divergent sequences can adopt a similar fold.     

Understanding relationship between sequence and functional evolution in yeast proteins

The underlying relationship between functional variables and sequence evolutionary rates is often assessed by partial correlation analysis. However, this strategy is impeded by the difficulty of conducting meaningful statistical analysis using noisy biological data. A recent study suggested that the partial correlation analysis is misleading when data is noisy and that the principal component regression analysis is a better tool to analyze biological data. In this paper, we evaluate how these two statistical tools (partial correlation and principal component regression) perform when data are noisy. Contrary to the earlier conclusion, we found that these two tools perform comparably in most cases. Furthermore, when there is more than one ‘true’ independent variable, partial correlation analysis delivers a better representation of the data. Employing both tools may provide a more complete and complementary representation of the real data. In this light, and with new analyses, we suggest that protein length and gene dispensability play significant, independent roles in yeast protein evolution. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.