Sequence of the phosphoenolpyruvate carboxykinase-encoding cDNA from the rumen anaerobic fungus Neocallimastix frontalis: comparison of the amino acid sequence with animals and yeast.

The nucleotide sequence of the cDNA of the phosphoenolpyruvate carboxykinase-encoding gene from the fungus Neocallimastix frontalis, was determined. The deduced amino acid sequence (608 residues) and the predicted protein structure were compared to their counterparts in animals and yeast. Catalytic regions (substrate-binding site and nucleotide-binding domains) are highly conserved among fungal and animal organisms. The yeast sequence showed no similarity to the fungal sequence.     

Integron integrases possess a unique additional domain necessary for activity.

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).     

Analysis of ancient sequence motifs in the H-PPase family

The unique family of membrane-bound proton-pumping inorganic pyrophosphatases, involving pyrophosphate as the alternative to ATP, was investigated by characterizing 166 members of the UniProtKB/Swiss-Prot + UniProtKB/TrEMBL databases and available completed genomes, using sequence comparisons and a hidden Markov model based upon a conserved 57-residue region in the loop between transmembrane segments 5 and 6. The hidden Markov model was also used to search the approximately one million sequences recently reported from a large-scale sequencing project of organisms in the Sargasso Sea, resulting in additional 164 partial pyrophosphatase sequences. The strongly conserved 57-residue region was found to contain two nonapeptidyl sequences, mainly consisting of the four 'very early' proteinaceous amino acid residues Gly, Ala, Val and Asp, compatible with an ancient origin of the inorganic pyrophosphatases. The nonapeptide patterns have charged amino acid residues at positions 1, 5 and 9, are apparent binding sites for the substrate and parts of the active site, and were shown to be so specific for these enzymes that they can be used for functional assignments of unannotated genomes.     

Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg²⁺-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80 Å resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg²⁺ accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function.     

Evolutionary conservation of the active site of soluble inorganic pyrophosphatase.

Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi). Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism. PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions. Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.     

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen-Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).     

Plant purine nucleotide cyclases

Cyclic nucleotides (cAMP and cGMP) play an essential role in many important cellular processes in prokaryotic and eukaryotic organisms. They are produced by purine nucleotide cyclases: adenylyl and guanylyl cyclases. They are classified as one of two distinct forms: soluble and bound to membranes. Beside the differences in enzyme localization, the domain structure and regulation of enzymes activity are also diverse. However, all cyclases possess three groups of important residues: substrate specifying residue, metal binding residues and transition state stabilization residues. The natural occurrence of cyclic nucleotides in plants is now established. It was shown that in higher plants cNMPs act as a second messengers in a large number of (patho)physiological responses. However, it is only recently that the first plant enzymes with AC and GC activity of the unique structure have been identified and functionally characterized. In this study a systematic analysis of all the known prokaryotic, fungal and animal cyclases was done and direct evidences for the presence AC and GC in plant cells were shown.     

<Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> has a family I pyrophosphatase: purification,cloning, and sequencing

The cytoplasmic pyrophosphatase of the photosynthetic bacterium Rhodospirillum rubrum was purified to electrophoretic homogeneity. The enzyme is a homohexamer of 20-kDa monomers. The gene was cloned and sequenced. Alignment of the deduced 179-amino-acid protein with known bacterial pyrophosphatases revealed conservation of all residues in the active site. Attempts to obtain an insertion mutant of the cytoplasmic pyrophosphatase gene did not yield any cell completely devoid of cytoplasmic pyrophosphatase activity. The mutants obtained showed 50% of the enzymatic activity and grew in twice the generation time of wild-type cells. This suggests that the membrane-bound pyrophosphatase of Rsp. rubrum is not sufficient for a normal growth rate, whereas the cytoplasmic enzyme is essential for growth. The characteristics of the gene and the encoded protein fit those of prokaryotic family I pyrophosphatases.     

The HopX (AvrPphE) family of Pseudomonas syringae type III effectors require a catalytic triad and a novel N-terminal domain for function

Many gram-negative plant pathogenic bacteria employ type III secretion systems to deliver effector proteins directly into the host cell during infection. On susceptible hosts, type III effectors aid pathogen growth by manipulating host defense pathways. On resistant hosts, some effectors can activate specific host disease resistance (R) genes, leading to generation of rapid and effective immune responses. The biochemical basis of these processes is poorly understood. The HopX (AvrPphE) family is a widespread type III effector among phytopathogenic bacteria. We determined that HopX family members are modular proteins composed of a conserved putative cysteine-based catalytic triad and a conserved potential target/cofactor interaction domain. HopX is soluble in host cells. Putative catalytic triad residues are required for avirulence activity on resistant bean hosts and for the generation of a cell-death response in specific Arabidopsis genotypes. The putative target/cofactor interaction domain is also required for these activities. Our data suggest that specific interaction with and modification of a cytosolic host target drives HopX recognition in resistant hosts and may contribute to virulence in susceptible hosts. Surprisingly, the Legionella pneumophila genome was found to contain a protein with similarity to HopX in sequence and domain arrangement, suggesting that these proteins might also contribute to animal pathogenesis and could be delivered to plant and animal hosts by diverse secretion systems.     

The oxygen binding site of cytochrome oxidase. Structural predictions on subunit I from amino acid sequences

Analyses of heme-attached amino acid sequences in known hemoprotein superfamilies provide a basis for prediction of such sequences in hemoproteins of unknown three-dimensional structure. Among 11 histidine residues conserved in subunit I of 3 mammalian and 2 fungal cytochrome oxidases the sequence around His-233 (human) is the most conserved and shows remarkable similarity to the sequence of the oxygen binding site in globins. Furthermore, the gene coding for subunit I in Saccharomyces cerevisiae and the gene for leghemoglobin in soybean are both split by introns right after these similar histidine sequences. The predicted distal histidine sequence of subunit I provides for heme a3 and Cua3 binding and has an extraordinarily high content of aromatic residues. These aromatic groups may serve as a molecular electron capacitor. Transmembrane sequences and electron transfer sequences are proposed.     

An ontology for cell types

We describe an ontology for cell types that covers the prokaryotic, fungal, animal and plant worlds. It includes over 680 cell types. These cell types are classified under several generic categories and are organized as a directed acyclic graph. The ontology is available in the formats adopted by the Open Biological Ontologies umbrella and is designed to be used in the context of model organism genome and other biological databases. The ontology is freely available at http://obo.sourceforge.net/ and can be viewed using standard ontology visualization tools such as OBO-Edit and COBrA.     

Computer modeling of two inorganic pyrophosphatases.

The yeast Saccharomyces cerevisiae has two inorganic pyrophosphatases that are structurally related. One, PPA1, is a cytoplasmic enzyme. The other, PPA2, is located in the mitochondria and appears to be energy-linked. The sequence similarity of PPA1 and PPA2 is about 66% and the identity is about 50%. All amino acids known to be important for catalysis are conserved, except one glutamate which is substituted by an aspartate in PPA2. The structures of PPA2 and the cytoplasmic PPase from Schizosaccharomyces pombe were modeled based on the three dimensional structure of PPA1. Two cysteines in PPA2 and one in the S. pombe enzyme are located at the catalytic cleft. Four residues form an unique insertion near the entrance of the catalytic cleft in the mitochondrial enzyme.     

H+-proton-pumping inorganic pyrophosphatase: a tightly membrane-bound family.

The earliest known H+-proton-pumping inorganic pyrophosphatase, the integrally membrane-bound H+-proton-pumping inorganic pyrophosphate synthase from Rhodospirillum rubrum, is still the only alternative to H+-ATP synthase in biological electron transport phosphorylation. Cloning of several higher plant vacuolar H+-proton-pumping inorganic pyrophosphatase genes has led to the recognition that the corresponding proteins form a family of extremely similar proton-pumping enzymes. The bacterial H+-proton-pumping inorganic pyrophosphate synthase and two algal vacuolar H+-proton-pumping inorganic pyrophosphatases are homologous with this family, as deduced from their cloned genes. The prokaryotic and algal homologues differ more than the H+-proton-pumping inorganic pyrophosphatases from higher plants, facilitating recognition of functionally significant entities. Primary structures of H+-proton-pumping inorganic pyrophosphatases are reviewed and compared with H+-ATPases and soluble proton-pumping inorganic pyrophosphatases.     

Mitochondrial complex I from Arabidopsis and rice: orthologs of mammalian and fungal components coupled with plant-specific subunits

The NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain is a large multisubunit complex in eukaryotes containing 30-40 different subunits. Analysis of this complex using blue-native gel electrophoresis coupled to tandem mass spectrometry (MS) has identified a series of 30 different proteins from the model dicot plant, Arabidopsis, and 24 different proteins from the model monocot plant, rice. These proteins have been linked back to genes from plant genome sequencing and comparison of this dataset made with predicted orthologs of complex I components in these plants. This analysis reveals that plants contain the series of 14 highly conserved complex I subunits found in other eukaryotic and related prokaryotic enzymes and a small set of 9 proteins widely found in eukaryotic complexes. A significant number of the proteins present in bovine complex I but absent from fungal complex I are also absent from plant complex I and are not encoded in plant genomes. A series of plant-specific nuclear-encoded complex I associated subunits were identified, including a series of ferripyochelin-binding protein-like subunits and a range of small proteins of unknown function. This represents a post-genomic and large-scale analysis of complex I composition in higher plants.     

Molecular evolution of thyroid peroxidase.

Thyroid peroxidase is a member of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and salivary peroxidase. Protein sequences showing a high degree of sequence similarity with mammalian peroxidases have recently been observed in several invertebrate species. A multiple sequence alignment prepared with five mammalian and six invertebrate peroxidases shows complete conservation of amino acid residues considered to be important in the formation of peroxidase compound 1. These include the distal and proximal histidines, a catalytic arginine residue, and an asparagine residue hydrogen bonded to the proximal histidine. TPO-2, an alternatively spliced form of TPO, lacks the essential asparagine (Asn 579). It is now possible to speak more broadly of the family of animal peroxidases, rather than mammalian peroxidases. The animal peroxidases comprise a group of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary and tertiary structure, but which share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relationship between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism.     

Mitochondrial complex I from Arabidopsis and rice: orthologs of mammalian and fungal components coupled with plant-specific subunits

The NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain is a large multisubunit complex in eukaryotes containing 30-40 different subunits. Analysis of this complex using blue-native gel electrophoresis coupled to tandem mass spectrometry (MS) has identified a series of 30 different proteins from the model dicot plant, Arabidopsis, and 24 different proteins from the model monocot plant, rice. These proteins have been linked back to genes from plant genome sequencing and comparison of this dataset made with predicted orthologs of complex I components in these plants. This analysis reveals that plants contain the series of 14 highly conserved complex I subunits found in other eukaryotic and related prokaryotic enzymes and a small set of 9 proteins widely found in eukaryotic complexes. A significant number of the proteins present in bovine complex I but absent from fungal complex I are also absent from plant complex I and are not encoded in plant genomes. A series of plant-specific nuclear-encoded complex I associated subunits were identified, including a series of ferripyochelin-binding protein-like subunits and a range of small proteins of unknown function. This represents a post-genomic and large-scale analysis of complex I composition in higher plants.     

Arabidopsis thaliana sequence analysis confirms the presenceof cyt b-561 in plants: Evidence for a novel protein family

Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented.     

Purifying selection and birth-and-death evolution in the class II hydrophobin gene families of the ascomycete <Emphasis Type="Italic">Trichoderma/Hypocrea</Emphasis>

Background  

Hydrophobins are proteins containing eight conserved cysteine residues that occur uniquely in mycelial fungi. Their main function is to confer hydrophobicity to fungal surfaces in contact with air or during attachment of hyphae to hydrophobic surfaces of hosts, symbiotic partners or themselves resulting in morphogenetic signals. Based on their hydropathy patterns and solubility characteristics, hydrophobins are divided into two classes (I and II), the latter being found only in ascomycetes.     

Cloning and sequencing of the gene for the cytoplasmic inorganic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum.

The gene (ppa) from the thermoacidophilic archaebacterium Thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned. Two degenerate oligonucleotide probes, synthesized according to the N-terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries. The DNA-derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms. The archaebacterial pyrophosphatase more closely resembles the eubacterial enzymes on the basis of sequence similarity and subunit size. The majority of amino acid residues considered to be essential for hydrolysis of pyrophosphate seem to have been conserved throughout evolution, as inferred from the results of an alignment of sequences from all three urkingdoms.     

Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae

UDP-glucose is the universal activated form of glucose, employed in all organisms for glucosyl transfer reactions and as precursor for various activated carbohydrates. In animal and fungal metabolism, UDP-glucose is required for utilization of galactose and for the synthesis of glycogen, the major carbohydrate storage polymer. The formation of UDP-glucose is catalyzed by UDP-glucose pyrophosphorylase (UGPase), which is highly conserved among eukaryotes. Here, we present the crystal structure of yeast UGPase, Ugp1p. Both in solution and in the crystal, Ugp1p forms homooctamers, which represent the enzymatically active form of the protein. Ugp1p subunits consist of three domains, with the active site presumably located in the central SpsA GnT I core (SGC) domain. The association in the octamer is mediated by contacts between left-handed beta-helices in the C-terminal domains, forming a toroidal solenoid structure in the core of the complex. The catalytic domains attached to this scaffold core do not directly contact each other, consistent with simple Michaelis-Menten kinetics found for Ugp1p. Conservation of hydrophobic residues at the subunit interfaces suggests that all fungal and animal homologs form this quarternary structure arrangement in contrast to monomeric plant UGPases, which have charged residues at these positions. Implications of this oligomeric arrangement for regulation of UGPase activity in fungi and animals are discussed.