SMART MODE:

NORMAL GENOMIC

• Cvrckova F
• Are plant formins integral membrane proteins?
• Genome Biol. 2000; 1: 1-1
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• BACKGROUND: The formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface. Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how. Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs. RESULTS: I found eight putative formin-coding genes in the publicly available part of the Arabidopsis genome sequence and analyzed their predicted protein sequences. Surprisingly, some of them lack parts of the conserved formin-homology 2 (FH2) domain and the majority of them seem to have signal sequences and putative transmembrane segments that are not found in yeast or animals formins. CONCLUSIONS: Plant formins define a distinct subfamily. The presence in most Arabidopsis formins of sequence motifs typical or transmembrane proteins suggests a mechanism of membrane attachment that may be specific to plant formins, and indicates an unexpected evolutionary flexibility of the conserved formin domain.

• Brugiere N, Rothstein SJ, Cui Y
• Molecular mechanisms of self-recognition in Brassica self-incompatibility.
• Trends Plant Sci. 2000; 5: 432-8
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• Plants have mechanisms to promote outbreeding and thereby to increase their genetic diversity. In species that are self-incompatible, self-pollen is rejected by the stigma. This mechanism has been the subject of intense study for many years and, in the past two years, significant progress has been made in identifying the genes involved in Brassica. Self-recognition involves two genes, one of which determines the male and the other the female specificity. Considerable progress has also been made on the mechanism by which self-recognition leads to pollen rejection, although the delineation of all the genes involved is still not complete.

• Faus I
• Recent developments in the characterization and biotechnological production of sweet-tasting proteins.
• Appl Microbiol Biotechnol. 2000; 53: 145-51
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• The state of the art regarding the six known sweet-tasting proteins (thaumatin, monellin, mabinlin, pentadin, brazzein and curculin) and the taste-modifying protein miraculin is reviewed. Their biochemical properties, molecular genetics and biotechnological production are assessed. All of these proteins have been isolated from plants that grow in tropical rainforests. They share no sequence homology or structural similarities. Nonetheless, one of them, thaumatin, shares extensive homology with certain non-sweet proteins found in other plants. The potential industrial applications of the sweet-tasting proteins are also discussed, placing special emphasis on the barriers that a recombinant product of these characteristics will have to overcome before it reaches the market.

• Eulgem T, Rushton PJ, Robatzek S, Somssich IE
• The WRKY superfamily of plant transcription factors.
• Trends Plant Sci. 2000; 5: 199-206
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• The WRKY proteins are a superfamily of transcription factors with up to 100 representatives in Arabidopsis. Family members appear to be involved in the regulation of various physio-logical programs that are unique to plants, including pathogen defense, senescence and trichome development. In spite of the strong conservation of their DNA-binding domain, the overall structures of WRKY proteins are highly divergent and can be categorized into distinct groups, which might reflect their different functions.

• Nagano Y
• Several features of the GT-factor trihelix domain resemble those of the Myb DNA-binding domain.
• Plant Physiol. 2000; 124: 491-4

• Pesce A et al.
• A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.
• EMBO J. 2000; 19: 2424-34
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• Small hemoproteins displaying amino acid sequences 20-40 residues shorter than (non-)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non-pathogenic unicellular organisms, and named 'truncated hemoglobins' (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a 'two-over-two' alpha-helical sandwich, reflecting an unprecedented editing of the classical 'three-over-three' alpha-helical globin fold. Based on specific Gly-Gly motifs the tertiary structure accommodates the deletion of the N-terminal A-helix and replacement of the crucial heme-binding F-helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

• Nesi N, Debeaujon I, Jond C, Pelletier G, Caboche M, Lepiniec L
• The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.
• Plant Cell. 2000; 12: 1863-78
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• The TRANSPARENT TESTA8 (TT8) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loop-helix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE (DFR) and BANYULS (BAN), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 (TTG1) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat.

• Juarez K, Flores H, Davila S, Olvera L, Gonzalez V, Morett E
• Reciprocal domain evolution within a transactivator in a restricted sequence space.
• Proc Natl Acad Sci U S A. 2000; 97: 3314-8
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• offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.

• Kitajima S, Koyama T, Ohme-Takagi M, Shinshi H, Sato F
• Characterization of gene expression of NsERFs, transcription factors of basic PR genes from Nicotiana sylvestris.
• Plant Cell Physiol. 2000; 41: 817-24
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• Three genes of NsERFs (EREBPs), transcription factors for GCC box of basic PR genes, were isolated from Nicotiana sylvestris. Analyses of transgenic tobacco carrying the NsERF promoter::GUS genes showed that expression of all NsERF genes in leaves was induced by ethylene. Sequence analyses indicated that the 5'-upstream region of NsERF3 gene has the GCC box. In contrast, the promoter regions of NsERF2 and 4 have no GCC box, whereas NsERF2 has a putative EIN3 binding site. Tissue/cell specific expression is also discussed.

• Rigden DJ, Mello LV, Bertioli DJ
• Structural modeling of a plant disease resistance gene product domain.
• Proteins. 2000; 41: 133-43
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• Dominant plant resistance genes are involved in the protection of plants against a wide variety of pathogens. Sequence analysis has revealed a variety of classes, often having domains in common. One commonly found region has come to be known as a putative nucleotide-binding site (NBS) due to the simple presence of sequence motifs. Until now, no experimental evidence has supported this idea. Here we suggest, as an alternative hypothesis, that part of this region is structurally homologous to the receiver domain common to many proteins of His-Asp phosphotransfer pathways. This conclusion is based on sequence analysis, threading experiments, and the construction of a molecular model of one domain that performs well against structure validation tools. The new hypothesis, in contrast to the NBS hypothesis, can explain the devastating effect of a Thr-->Ala mutation in a well-characterized resistance gene product. According to the new hypothesis, regions located N-terminal and C-terminal to the modeled portion, containing highly conserved sequence motifs, could form a separate domain.

• Zhang Z, Hong Z, Verma DP
• Phragmoplastin polymerizes into spiral coiled structures via intermolecular interaction of two self-assembly domains.
• J Biol Chem. 2000; 275: 8779-84
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• Phragmoplastin, a high molecular weight GTPase belonging to the dynamin superfamily of proteins, becomes associated with the cell plate during cytokinesis in plants. Growth of the cell plate requires continuous fusion of vesicles, and phragmoplastin appears to play a role in the formation of vesicle-tubule-vesicle structures at the cell plate. In this study, we have demonstrated that two self-assembly domains (SA1 and SA2) are involved in polymerization of phragmoplastin. SA1 is about 42 amino acids long and is located near the N terminus overlapping with the GTP-binding region. SA2, containing at least 24 amino acids, is located in the middle of the molecule outside the GTP-binding domain. Peptides containing either SA1 or SA2 interact efficiently with the full-length phragmoplastin. The SA1 domain of one phragmoplastin molecule also binds to SA2 of another as confirmed in vitro by using radiolabeled peptides. This interaction leads to the formation of polymers with a staggered contoured spiral structure. Electron microscopy studies revealed that helical arrays of phragmoplastin can be induced by reducing salt concentration. Our results suggest that phragmoplastin may assemble into helical arrays that wrap around and squeeze vesicles into vesicle-tubule-vesicle structures observed on the forming cell plate.

• May T, Soll J
• 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants.
• Plant Cell. 2000; 12: 53-64
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• Transit sequences of chloroplast-destined precursor proteins are phosphorylated on a serine or threonine residue. The amino acid motif around the phosphorylation site is related to the phosphopeptide binding motif for 14-3-3 proteins. Plant 14-3-3 proteins interact specifically with wheat germ lysate-synthesized chloroplast precursor proteins and require an intact phosphorylation motif within the transit sequence. Chloroplast precursor proteins do not interact with 14-3-3 when synthesized in the heterologous reticulocyte lysate. In contrast, a precursor protein destined for plant mitochondria was found to be associated with 14-3-3 proteins present in the reticulocyte lysate but not with 14-3-3 from wheat germ lysate. This indicates an unrecognized selectivity of 14-3-3 proteins for precursors from mitochondria and plastids in plants in comparison to fungi and animals. The heterooligomeric complex has an apparent size of 200 kD. In addition to the precursor protein, it contains 14-3-3 (probably as a dimer) and a heat shock protein Hsp70 isoform. Dissociation of the precursor complex requires ATP. Protein import experiments of precursor from the oligomeric complex into intact pea chloroplasts reveal three- to fourfold higher translocation rates compared with the free precursor, which is not complexed. We conclude that the 14-3-3-Hsp70-precursor protein complex is a bona fide intermediate in the in vivo protein import pathway in plants.

• Pan Q, Wendel J, Fluhr R
• Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes.
• J Mol Evol. 2000; 50: 203-13
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• The majority of plant disease resistance genes are members of very large multigene families. They encode structurally related proteins containing nucleotide binding site domains (NBS) and C-terminal leucine rich repeats (LRR). The N-terminal region of some resistance genes contain a short sequence called TIR with homology to the animal innate immunity factors, Toll and interleukin receptor-like genes. Only a few plant resistance genes have been functionally analyzed and the origin and evolution of plant resistance genes remain obscure. We have reconstructed gene phylogeny by exhaustive analysis of available genome and amplified NBS domain sequences. Our study shows that NBS domains faithfully predict whole gene structure and can be divided into two major groups. Group I NBS domains contain group-specific motifs that are always linked with the TIR sequence in the N terminus. Significantly, Group I NBS domains and their associated TIR domains are widely distributed in dicot species but were not detected in cereal databases. Furthermore, Group I specific NBS sequences were readily amplified from dicot genomic DNA but could not be amplified from cereal genomic DNA. In contrast, Group II NBS domains are always associated with putative coiled-coil domains in their N terminus and appear to be present throughout the angiosperms. These results suggest that the two main groups of resistance genes underwent divergent evolution in cereal and dicot genomes and imply that their cognate signaling pathways have diverged as well.

• Jakobsson PJ, Morgenstern R, Mancini J, Ford-Hutchinson A, Persson B
• Membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). A widespread protein superfamily.
• Am J Respir Crit Care Med. 2000; 161: 204-204
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• The members of the MAPEG superfamily have been aligned and found to be distantly related, with a common pattern of hydropathy. Figure 2A shows the multiple sequence alignments of the human members and Figure 2B the corresponding superimposed hydropathy profiles. The alignment in Figure 2A demonstrates a total of six strictly conserved residues. The Arg-51 in LTC4 synthase has been suggested to function as proton donor for the opening of the LTA4 epoxide. This arginine is found in all but the FLAP sequences in accordance with the observation that FLAP has no known enzyme activity. Also the Tyr-93 in LTC4 synthase has been suggested to function as a base for the formation of the thiolate anion of glutathione. This tyrosine is not conserved in MGST1 or MGST1-L1. Table 1 summarizes some other properties of the individual human proteins. They are all of the same size, ranging from 147 to 161 amino acids. Only FLAP differs in that its isoelectric point is more neutral than that of the other, more basic proteins. The genes encoding these proteins all reside on different chromosomes (when known) (Table 1). In addition to the human proteins, MAPEG members have been identified in plants, fungi, and bacteria. It is clearly a challenge to elucidate their role in these different phyla in relation to their defined physiological functions in humans.

• Yu HG, Hiatt EN, Dawe RK
• The plant kinetochore.
• Trends Plant Sci. 2000; 5: 543-7
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• Kinetochores are large protein complexes that bind to centromeres. By interacting with microtubules and their associated motor proteins, kinetochores both generate and regulate chromosome movement. Kinetochores also function in the spindle checkpoint; a surveillance mechanism that ensures that metaphase is complete before anaphase begins. Although the ultrastructure of plant kinetochores has been known for many years, only recently have specific kinetochore proteins been identified. The recent data indicate that plant kinetochores contain homologs of many of the proteins implicated in animal and fungal kinetochore function, and that the plant kinetochore is a redundant structure with distinct biochemical subdomains.

• Chakraborty S et al.
• Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II.
• Protein Eng. 2000; 13: 551-5
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• Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.

• van Aalten DM et al.
• Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-A resolution.
• Proc Natl Acad Sci U S A. 2000; 97: 5842-7
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• In this paper, we describe the structure of chitinase B from Serratia marcescens, which consists of a catalytic domain with a TIM-barrel fold and a 49-residue C-terminal chitin-binding domain. This chitinase is the first structure of a bacterial exochitinase, and it represents one of only a few examples of a glycosyl hydrolase structure having interacting catalytic and substrate-binding domains. The chitin-binding domain has exposed aromatic residues that contribute to a 55-A long continuous aromatic stretch extending into the active site. Binding of chitin oligomers is blocked beyond the -3 subsite, which explains why the enzyme has chitotriosidase activity and degrades the chitin chain from the nonreducing end. Comparison of the chitinase B structure with that of chitinase A explains why these enzymes act synergistically in the degradation of chitin.

• Small ID, Peeters N
• The PPR motif - a TPR-related motif prevalent in plant organellar proteins.
• Trends Biochem Sci. 2000; 25: 46-7

• Noiraud N, Delrot S, Lemoine R
• The sucrose transporter of celery. Identification and expression during salt stress.
• Plant Physiol. 2000; 122: 1447-55
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• In celery (Apium graveolens L.), long-distance transport of reduced carbon occurs both in the form of sucrose (Suc) and mannitol. The presence of mannitol has been related to the resistance of celery to salt stress. To investigate the transport events occurring during salt stress, we have cloned the H(+)/Suc transporter of celery AgSUT1 (A. graveolens Suc uptake transport 1) from a mature leaf cDNA library. The function of the encoded protein was confirmed by expression in yeast. AgSUT1 is a H(+)/Suc transporter with a high affinity for Suc (K(m) of 139 microM). Another closely related cDNA (AgSUT2) was also identified. AgSUT1 is mainly expressed in mature leaves and phloem of petioles, but also in sink organs such as roots. When celery plants were subjected to salt stress conditions (30 d watering with 300 mM NaCl) favoring mannitol accumulation (J.D. Everard, R. Gucci, S.C. Kann, J.A. Flore, W.H. Loescher [1994] Plant Physiol 106: 281-292), AgSUT1 expression was decreased in all organs, but markedly in roots. The results are discussed in relation to the physiology of celery.

• Savino C, Federici L, Ippoliti R, Lendaro E, Tsernoglou D
• The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome.
• FEBS Lett. 2000; 470: 239-43
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• The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.

• Liu Y et al.
• Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from pokeweed.
• Plant Physiol. 2000; 122: 1015-24
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• A small cysteine-rich protein with antimicrobial activity was isolated from pokeweed (Phytolacca americana) seeds and purified to homogeneity. The protein inhibits the growth of several filamentous fungi and gram-positive bacteria. The protein was highly basic, with a pI higher than 10. The entire amino acid sequence of the protein was determined to be homologous to antimicrobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. americana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and sequenced. The deduced amino acid sequence shows the presence of a signal peptide at the amino terminus, suggesting that the protein is synthesized as a preprotein and secreted outside the cells. The chromosomal gene shows the presence of an intron located within the region encoding the signal peptide. Southern hybridization showed that there was small gene family encoding Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, and was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted into the environment of the seeds during germination, and may create an inhibitory zone against soil-borne microorganisms. The disulfide bridges of Pa-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicates that the protein has a small cystine-knot folding, a positive patch, and a hydrophobic patch.

• Jones HD, Kurup S, Peters NC, Holdsworth MJ
• Identification and analysis of proteins that interact with the Avena fatua homologue of the maize transcription factor VIVIPAROUS 1.
• Plant J. 2000; 21: 133-42
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• The Avena fatua (wild oat) homologue of VIVIPAROUS 1 (AfVP1) has been implicated in controlling the maintenance of embryo dormancy in mature imbibed seeds, but the detailed mechanisms by which this transcription factor family activates embryo maturation pathways and simultaneously represses germination are not known. A two-hybrid screen in yeast identified three proteins that interacted specifically with AfVP1 (AfVP1 interacting proteins; AfVIPs). AfVIPs 2 and 3 interacted with the C-terminus of AfVP1, which contains the B2 + B3 domains, previously shown to bind DNA, whereas AfVIP1 interacted with the isolated B3 domain. Using purified proteins in in vitro experiments, all three AfVIPs were shown also to interact with the Arabidopsis homologue ABSCISIC ACID INSENSITIVE 3 (ABI3). The three AfVIPs were expressed in both dormant and non-dormant embryos, but the abundance of AfVIP1 and 3 transcripts was greater in germinated than dormant seeds, whereas transcripts of AfVIP2 (and AfVP1) were more highly expressed in dormant embryos. The AfVIP3 protein has homology to a human cell-crisis gene with a predicted role in the cell cycle; AfVIP2 contains a ring-type zinc finger motif. These homologies, together with analysis of expression studies, suggest that these proteins may play specific roles in AfVP1-mediated regulation of the dormancy to germination transition in A. fatua seeds.

• Kranz H, Scholz K, Weisshaar B
• c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages.
• Plant J. 2000; 21: 231-5
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• Since the identification of the first plant MYB-like protein, the Zea mays factor C1, the number of MYB-related genes described has greatly increased. All of the more than 150 plant MYB-like proteins known so far contain either two or only one sequence-related helix-turn-helix motif in their DNA-binding domain. Animal c-MYB genes contain three such helix-turn-helix motif-encoding repeats (R1R2R3 class genes). It has therefore been concluded that R2R3-MYB genes are the plant equivalents of c-MYB and that there are significant differences in the basic structure of MYB genes of plants and animals. Here, we describe expressed R1R2R3-MYB genes from Physcomitrella patients++ and Arabidopsis thaliana, designated PpMYB3R-1 and AtMYB3R-1. The amino acid sequences of their DNA-binding domains show high similarity to those of animal MYB factors, and less similarity to R2R3-MYB proteins from plants. In addition, R1R2R3-MYB genes were identified in different plant evolutionary lineages including mosses, ferns and monocots. Our data show that a DNA-binding domain consisting of three MYB repeats existed before the divergence of the animal and plant lineages. R1R2R3-MYB genes may have a conserved function in eukaryotes, and R2R3-MYB genes may predominantly regulate plant-specific processes which evolved during plant speciation.

• Moraes RA et al.
• Lima bean (Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea weevil (Callosobruchus maculatus).
• Braz J Med Biol Res. 2000; 33: 191-8
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• The presence of phaseolin (a vicilin-like 7S storage globulin) peptides in the seed coat of the legume Phaseolus lunatus L. (lima bean) was demonstrated by N-terminal amino acid sequencing. Utilizing an artificial seed system assay we showed that phaseolin, isolated from both cotyledon and testa tissues of P. lunatus, is detrimental to the nonhost bruchid Callosobruchus maculatus (F) (cowpea weevil) with ED50 of 1.7 and 3.5%, respectively. The level of phaseolin in the seed coat (16.7%) was found to be sufficient to deter larval development of this bruchid. The expression of a C. maculatus-detrimental protein in the testa of nonhost seeds suggests that the protein may have played a significant role in the evolutionary adaptation of bruchids to legume seeds.

• Beekwilder J, Schipper B, Bakker P, Bosch D, Jongsma M
• Characterization of potato proteinase inhibitor II reactive site mutants.
• Eur J Biochem. 2000; 267: 1975-84
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• Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nM) and chymotrypsin (Ki = 0.9 nM), and is quite robust towards mutations at positions P2 or P1'. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nM), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.

• Williams L, Grafi G
• The retinoblastoma protein - a bridge to heterochromatin.
• Trends Plant Sci. 2000; 5: 239-40

• van der Knaap E, Kim JH, Kende H
• A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth.
• Plant Physiol. 2000; 122: 695-704
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• Os-GRF1 (Oryza sativa-GROWTH-REGULATING FACTOR1) was identified in a search for genes that are differentially expressed in the intercalary meristem of deepwater rice (Oryza sativa L.) internodes in response to gibberellin (GA). Os-GRF1 displays general features of transcription factors, contains a functional nuclear localization signal, and has three regions with similarities to sequences in the database. One of these regions is similar to a protein interaction domain of SWI2/SNF2, which is a subunit of a chromatin-remodeling complex in yeast. The two other domains are novel and found only in plant proteins of unknown function. To study its role in plant growth, Os-GRF1 was expressed in Arabidopsis. Stem elongation of transformed plants was severely inhibited, and normal growth could not be recovered by the application of GA. Our results indicate that Os-GRF1 belongs to a novel class of plant proteins and may play a regulatory role in GA-induced stem elongation.

• Barciszewski J, Szymanski M, Haertle T
• Minireview: analysis of rape seed napin structure and potential roles of the storage protein.
• J Protein Chem. 2000; 19: 249-54
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• Structural and functional data on 2S albumins and particularly rape seed napins are reviewed and, based on the coordinates of the three-dimensional structure of napin-like albumin BnIb, are used to model different rape napins. Surprisingly, the modeled napins, despite great sequence homology, differ in tertiary arrangements of the polypeptide chains. It is proposed that these differences in 3D structures of the analyzed rape napins may reflect their functions, which may cover many other potential beneficial purposes besides simple storage.

• Flower DR, North AC, Sansom CE
• The lipocalin protein family: structural and sequence overview.
• Biochim Biophys Acta. 2000; 1482: 9-24
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• Lipocalins are remarkably diverse at the sequence level yet have highly conserved structures. Most lipocalins share three characteristic conserved sequence motifs - the kernel lipocalins - while others are more divergent family members - the outlier lipocalins - typically sharing only one or two. This classification is a useful tool for analysing the family, and within these large sets are smaller groups sharing much higher levels of sequence similarity. The lipocalins are also part of a larger protein superfamily: the calycins, which includes the fatty acid binding proteins, avidins, a group of metalloproteinase inhibitors, and triabin. The superfamily is characterised by a similar structure (a repeated +1 topology beta-barrel) and by the conservation of a remarkable structural signature.

• Alvarez-Buylla ER et al.
• An ancestral MADS-box gene duplication occurred before the divergence of plants and animals.
• Proc Natl Acad Sci U S A. 2000; 97: 5328-33
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• Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

• Hadlington JL, Denecke J
• Sorting of soluble proteins in the secretory pathway of plants.
• Curr Opin Plant Biol. 2000; 3: 461-8
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• The secretory pathway of plants is a network of organelles that communicate via vesicle transport. This process involves budding on donor membranes followed by their targeting to, recognition by and fusion with the acceptor membrane. Protein sorting through the plant secretory pathway is a process that requires the specific recognition of signals by receptor molecules. For soluble proteins, recognition takes place in the lumen of the secretory pathway. The sorting receptors must mediate signal transduction across the membrane to convey the information about the presence of cargo molecules to cytosolic factors, which regulate the formation of transport vesicles. Recently, a number of key elements in this process have been identified, providing tools to study protein sorting at the molecular level.

• Paolocci F, Capucci R, Arcioni S, Damiani F
• Birdsfoot trefoil: a model for studying the synthesis of condensed tannins.
• Basic Life Sci. 1999; 66: 343-56

• Stacy RA, Nordeng TW, Culianez-Macia FA, Aalen RB
• The dormancy-related peroxiredoxin anti-oxidant, PER1, is localized to the nucleus of barley embryo and aleurone cells.
• Plant J. 1999; 19: 1-8
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• Protection against desiccation-induced injury, including damage by reactive oxygen species (ROS), is a necessary component of the genetic programmes active during late seed development. Likewise, protection against ROS respiration by-products is required during seed imbibition and germination. Late embryogenesis abundant (LEA) proteins are proposed to protect seed tissues against desiccation-induced damage. Specifically, the atypical Lea gene Per1 in barley (Hordeum vulgare L.) has been proposed to play a protective role in embryo and aleurone cells against free-radical damage during late seed development and early imbibition. PER1 represents a subgroup of the peroxiredoxin family of thiol-requiring anti-oxidants with one conserved cysteine residue (1-Cys), and displays in vitro anti-oxidant activity. In this work, we use antiserum generated against PER1 to study protein accumulation patterns as well as localization at the tissue, cellular and subcellular level. While previous studies have shown the Per1 transcript to be dormancy-related, we show here that the protein level is maintained in imbibed dormant seeds, but not in non-dormant seeds. Our data identify the location of this seed-specific peroxiredoxin as the nucleus of immature embryos and aleurone layers. Highest levels of protein are detected in nucleoli. In contrast, in mature imbibed dormant seeds, cytosolic levels are comparable to that of the nucleus. A putative nuclear localization signal (NLS) of bipartite nature was identified in the C-terminal end of the PER1 sequence. Protective roles for PER1 in seeds are discussed.

• Schaller A
• Oligopeptide signalling and the action of systemin.
• Plant Mol Biol. 1999; 40: 763-9

• Mushegian A
• The minimal genome concept.
• Curr Opin Genet Dev. 1999; 9: 709-14
• Display abstract

• Complete genome sequences are becoming available for a large number of diverse species. Quantification of gene content, of gene family expansion, of orthologous gene conservation, as well as their displacement, are now possible - laying the ground for the estimation of the minimal set of proteins sufficient for cellular life. The consensus of computational results suggests a set close to 300 genes. These predictions will be evaluated by engineering of small bacterial genomes.

• McManus AM et al.
• MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.
• J Mol Biol. 1999; 293: 629-38
• Display abstract

• MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides.The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel.This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.

• Felix G, Duran JD, Volko S, Boller T
• Plants have a sensitive perception system for the most conserved domain of bacterial flagellin.
• Plant J. 1999; 18: 265-76
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• The flagellum is an important virulence factor for bacteria pathogenic to animals and plants. Here we demonstrate that plants have a highly sensitive chemoperception system for eubacterial flagellins, specifically targeted to the most highly conserved domain within its N terminus. Synthetic peptides comprising 15-22 amino acids of this domain acted as elicitors of defence responses at sub-nanomolar concentrations in cells of tomato and several other plant species. Peptides comprising only the central 8 to 11 amino acids of the active domain had no elicitor activity but acted as specific, competitive inhibitors in tomato cells. These antagonists suppressed the plant's response to flagellin, crude bacterial extracts and living bacterial cells. Thus, plants have a highly sensitive and selective perception system for the flagellin of motile eubacteria.

• Grenier J, Potvin C, Trudel J, Asselin A
• Some thaumatin-like proteins hydrolyse polymeric beta-1,3-glucans.
• Plant J. 1999; 19: 473-80
• Display abstract

• Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.

• Domingo C, Sauri A, Mansilla E, Conejero V, Vera P
• Identification of a novel peptide motif that mediates cross-linking of proteins to cell walls.
• Plant J. 1999; 20: 563-70
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• A cDNA clone representing a member of a novel class of cell wall proteins was isolated from tobacco plants. We have designated this protein NtTLRP for tyrosine- and lysine-rich protein. It is structurally related to the previously identified TLRP from tomato plants, sharing a high amino-acid sequence similarity at the C-terminal region. This region contains what appears to be a novel peptide motif which we call CD for cysteine-rich domain, and which is common to several other cell-wall proteins. By using a functional test in transgenic plants, we demonstrate that the presence of the CD domain is per se sufficient to cross-link previously soluble proteins to the cell wall. We present evidence that NtTLRP is cross-linked and specifically localizes to the cell wall of lignified cells. The highly localized deposition of NtTLRP in these cells indicates that this class of cell-wall proteins may have a specialized function in the formation of xylem tissue.

• Dodeman VL, Le Guilloux M, Ducreux G, de Vienne D
• Characterization of storage proteins in Daucus carota L.: two novel proteins display zygotic embryo specificity.
• Plant Cell Physiol. 1998; 39: 49-56
• Display abstract

• Although maturation-related proteins are well known in the endosperm of albuminous seeds, an important question is whether the zygotic embryo possesses its own maturation proteins. We report on the isolation and partial characterization of storage proteins of carrot (Daucus carota L. var Nandor) dry achenes and isolated zygotic embryos, using one- and two-dimensional electrophoresis techniques, HPLC and amino acid sequencing. The presence of a series of abundant polypeptides showing charge heterogeneity, that are rapidly degraded upon germination, was revealed in the endosperm. These proteins consisted of glycoproteins, the most abundant of which displayed a molecular mass (M(r)) of 58,000, albumins of M(r) 42,000 comprising at least one beta-1,3-glucanase, and two globulins of M(r) 90,000 and 50,000-55,000 respectively, the second being an oligomer composed of three subunits of M(r) 13,000, 20,000 and 30,000. None of these storage proteins identified in the endosperm were detected in zygotic embryos. In contrast, two novel proteins were isolated from zygotic embryos, namely a globulin family of M(r) 50,000 and pI 6.3-6.8, which was named "daucin", and a late embryogenesis abundant (LEA) protein family of M(r) 25,000 and pI 6.3-6.6, named "RAB25". Since the latter proteins are apparently absent of the endosperm, these results suggest that the maturation of carrot zygotic embryos requires its own specific set of storage and LEA proteins.

• Shutov AD, Braun H, Chesnokov YV, Horstmann C, Kakhovskaya IA, Baumlein H
• Sequence peculiarity of gnetalean legumin-like seed storage proteins.
• J Mol Evol. 1998; 47: 486-92
• Display abstract

• The development of seeds as a specialized organ for the nutrition, protection, and dispersal of the next generation was an important step in the evolution of land plants. Seed maturation is accompanied by massive synthesis of storage compounds such as proteins, starch, and lipids. To study the processes of seed storage protein evolution we have partially sequenced storage proteins from maturing seeds of representatives from the gymnosperm genera Gnetum, Ephedra, and Welwitschia-morphologically diverse and unusual taxa that are grouped in most formal systems into the common order Gnetales. Based on partial N-terminal amino acid sequences, oligonucleotide primers were derived and used for PCR amplification and cloning of the corresponding cDNAs. We also describe the structure of the nuclear gene for legumin of Welwitschia mirabilis. This first gnetalean nuclear gene structure contains introns in only two of the four conserved positions previously characterized in other spermatophyte legumin genes. The distinct phylogenetic status of the gnetalean taxa is also reflected in a sequence peculiarity of their legumin genes. A comparative analysis of exon/intron sequences leads to the hypothesis that legumin genes from Gnetales belong to a monophyletic evolutionary branch clearly distinct from that of legumin genes of extant Ginkgoales and Coniferales as well as from all angiosperms.

• Jensen SO, Reeves PR
• Domain organisation in phosphomannose isomerases (types I and II).
• Biochim Biophys Acta. 1998; 1382: 5-7
• Display abstract

• Phosphomannose isomerase (PMI) types I and II were found to possess a conserved protein motif. This motif coincides with the catalytic site of the Candida albicans type I PMI, indicating a common catalytic process for both PMI types. The type II PMI are bifunctional enzymes possessing PMI and guanosine diphospho-D-mannose pyrophosphorylase (GMP) activity in separate catalytic domains, which in some species may function as separate proteins.

• Koo JC et al.
• Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity.
• Biochim Biophys Acta. 1998; 1382: 80-90
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• Two antifungal peptides (Pn-AMP1 and Pn-AMP2) have been purified to homogeneity from seeds of Pharbitis nil. The amino acid sequences of Pn-AMP1 (41 amino acid0 residues) and Pn-AMP2 (40 amino acid residues) were identical except that Pn-AMP1 has an additional serine residue at the carboxyl-terminus. The molecular masses of Pn-AMP1 and Pn-AMP2 were confirmed as 4299.7 and 4213.2 Da, respectively. Both the Pn-AMPs were highly basic (pI 12.02) and had characteristics of cysteine/glycine rich chitin-binding domain. Pn-AMPs exhibited potent antifungal activity against both chitin-containing and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/ml for Pn-AMP1 and from 0.6 to 75 micrograms/ml for Pn-AMP2. The Pn-AMPs penetrated very rapidly into fungal hyphae and localized at septum and hyphal tips of fungi, which caused burst of hyphal tips. Burst of hyphae resulted in disruption of the fungal membrane and leakage of the cytoplasmic materials. To our knowledge, Pn-AMPs are the first hevein-like proteins that show similar fungicidal effects as thionins do.

• Helleboid S, Bauw G, Belingheri L, Vasseur J, Hilbert JL
• Extracellular beta-1,3-glucanases are induced during early somatic embryogenesis in Cichorium.
• Planta. 1998; 205: 56-63
• Display abstract

• In leaf tissues of the Cichorium hybrid clone '474' (C. intybus L. var. sativum x C. endivia L. var. latifolia), the acquisition and expression of embryogenic competence was characterised by the appearance of 15 polypeptides (Boyer et al., 1993, Plant Sci 93: 41-53). The 38-kDa proteins were found to be abundantly present in conditioned embryogenic medium after the first division of the induced cells. These proteins seemed to be glycosylated as indicated by general carbohydrate detection methods. Internal amino-acid sequences obtained after microsequencing tryptic peptides appeared to be 36-57% homologous with plant beta-1,3-endoglucanases. In addition, these 38-kDa proteins were recognised by antibodies raised against the pathogenesis-related tobacco glucanase PR2a and their beta-1,3-glucanase activity was demonstrated by direct detection in polyacrylamide gels after electrophoresis. These results strongly suggested that the 38-kDa somatic-embryogenesis-related (SER) polypeptides are beta-1,3-glucanases. Moreover, the level of glucanase activity was nearly three times higher in the medium of the embryogenic '474' line than in the medium of a non-embryogenic line. The possible involvement of the extracellular 38-kDa proteins in callose degradation during somatic embryogenesis is discussed.

• Dunwell JM
• Cupins: a new superfamily of functionally diverse proteins that include germins and plant storage proteins.
• Biotechnol Genet Eng Rev. 1998; 15: 1-32

• Dunwell JM
• Sequence analysis of the cupin gene family in Synechocystis PCC6803.
• Microb Comp Genomics. 1998; 3: 141-8
• Display abstract

• The recently described cupin superfamily of proteins includes the germin and germinlike proteins, of which the cereal oxalate oxidase is the best characterized. This superfamily also includes seed storage proteins, in addition to several microbial enzymes and proteins with unknown function. All these proteins are characterized by the conservation of two central motifs, usually containing two or three histidine residues presumed to be involved with metal binding in the catalytic active site. The present study on the coding regions of Synechocystis PCC6803 identifies a previously unknown group of 12 related cupins, each containing the characteristic two-motif signature. This group comprises 11 single-domain proteins, ranging in length from 104 to 289 residues, and includes two phosphomannose isomerases and two epimerases involved in cell wall synthesis, a member of the pirin group of nuclear proteins, a possible transcriptional regulator, and a close relative of a cytochrome c551 from Rhodococcus. Additionally, there is a duplicated, two-domain protein that has close similarity to an oxalate decarboxylase from the fungus Collybia velutipes and that is a putative progenitor of the storage proteins of land plants.

• Stacy RA, Aalen RB
• Identification of sequence homology between the internal hydrophilic repeated motifs of group 1 late-embryogenesis-abundant proteins in plants and hydrophilic repeats of the general stress protein GsiB of Bacillus subtilis.
• Planta. 1998; 206: 476-8
• Display abstract

• Late embryogenesis abundant (LEA) proteins are speculated to protect against water stress in plants. Group 1 LEA proteins are hydrophilic and vary mainly in the numbers of an extremely hydrophilic internal 20-amino-acid motif. This motif is present up to four times in Arabidopsis thaliana and Hordeum vulgare Group 1 proteins and has been described in numerous plant species. However, no similarity has yet been described between Group 1 genes or gene products and those from non-plant species. We report here the striking similarity between the repeated internal motif of Group 1 LEA proteins and a repeated hydrophilic motif present in a stress-related protein (GsiB) from Bacillus subtilis.

• Mian IS
• Sequence, structural, functional, and phylogenetic analyses of three glycosidase families.
• Blood Cells Mol Dis. 1998; 24: 83-100
• Display abstract

• Glycosidases, which cleave the glycosidic bond between a carbohydrate and another moiety, have been classified into over 63 families. Here, a variety of computational techniques have been employed to examine three families important in normal and abnormal pathology with the aim of developing a framework for future homology modeling, experimental and other studies. Family 1 includes bacterial and archaeal enzymes as well as lactase phlorizin-hydrolase and klotho, glycosidases implicated in disaccharide intolerance II and aging respectively. A statistical model, a hidden Markov model (HMM), for the family 1 glycosidase domain was trained and used as the basis for comparative examination of the conserved and variable sequence and structural features as well as the phylogenetic relationships between family members. Although the structures of four family 1 glycosidases have been determined, this is the first comparative examination of all these enzymes. Aspects that are unique to specific members or subfamilies (substrate binding loops) as well those common to all members (a beta/alpha)8 barrel fold) have been defined. Active site residues in some domains in klotho and lactase-phlorizin hydrolases differ from other members and in one instance may bind but not cleave substrate. The four invariant and most highly conserved residues are not residues implicated in catalysis and/or substrate binding. Of these, a histidine may be involved in transition state stabilization. Glucosylceramidase (family 30) and galactosylceramidase (family 59) are mutated in the lysosomal storage disorders Gaucher disease and Krabbe disease, respectively. HMM-based analysis, structure prediction studies and examination of disease mutations reveal a glycosidase domain common to these two families that also occurs in some bacterial glycosidases. Similarities in the reactions catalyzed by families 30 and 59 are reflected in the presence of a structurally and functionally related (beta/alpha)8 barrel fold related to that in family 1.

• Shutov AD, Braun H, Chesnokov YV, Baumlein H
• A gene encoding a vicilin-like protein is specifically expressed in fern spores. Evolutionary pathway of seed storage globulins.
• Eur J Biochem. 1998; 252: 79-89
• Display abstract

• The isolation and characterisation of a cDNA coding for a vicilin-like protein of the fern Matteuccia struthiopteris is described. The corresponding gene is specifically expressed during late stages of spore development. Extensive sequence comparisons suggest that the fern protein can be considered as a molecular missing link between single-domain germin/spherulin-like proteins and two-domain seed storage globulins of gymnosperms and angiosperms. Further, evidence is provided for the existence of a superfamily of structurally related, functionally different proteins which includes storage globulins of the vicilin and legumin families, a membrane-associated sucrose-binding protein of soybean, a Forssman antigen-binding lectin of velvet bean, the precursor of the vacuolar membrane bound proteins MP27/MP32 of pumpkin, the embryogenesis-specific protein Gea8 of carrot, the fern-spore-specific protein described here as well as the functionally diverse family of germins/germin-like proteins and the spherulins of myxomycetes. We propose that seed storage globulins of spermatophytes evolved from desiccation-related single-domain proteins of prokaryotes via a duplicated two-domain ancestor that is best represented by the extant fern spore-specific vicilin-like protein.

• Hattori J, Boutilier KA, van Lookeren Campagne MM, Miki BL
• A conserved BURP domain defines a novel group of plant proteins with unusual primary structures.
• Mol Gen Genet. 1998; 259: 424-8
• Display abstract

• We have identified a new class of plant proteins containing a common C-terminal region, which we have termed the BURP domain. These proteins are defined not only by the BURP domain, but also by the overall similarity in their modular construction. The BURP domain proteins consist of either three or four modules: (i) an N-terminal hydrophobic domain -- a presumptive transit peptide, joined to (ii) a short conserved segment or other short segment, (iii) an optional segment consisting of repeated units which is unique to each member, and (iv) the C-terminal BURP domain. These individual modules appear to be combined to form two main classes of BURP domain proteins. The BURP domain proteins, despite the similarities in their primary structural features, show no obvious similarities in the tissues or conditions under which they are expressed. The presence of the conserved BURP domain in diverse plant proteins suggests an important and fundamental functional role for this domain.

• Muntz K
• Deposition of storage proteins.
• Plant Mol Biol. 1998; 38: 77-99
• Display abstract

• Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.

• Gane PJ, Dunwell JM, Warwicker J
• Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase.
• J Mol Evol. 1998; 46: 488-93
• Display abstract

• It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved beta-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the beta-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase.

• Ishihara H, Sasagawa T, Sakai R, Nishikawa M, Kimura M, Funatsu G
• Isolation and molecular characterization of four arginine/glutamate rich polypeptides from the seeds of sponge gourd (Luffa cylindrica).
• Biosci Biotechnol Biochem. 1997; 61: 168-70
• Display abstract

• Four arginine/glutamate rich polypeptides referred to as 5 k-, 6.5 k-, 12.5 k-, and 14 k-AGRPs were purified to homogeneity by gel filtration on Sephadex G-75 followed by CM-cellulose, butyl-Toyopearl 650 M, and reverse-phase HPLC from the seed of sponge gourd (Luffa cylindrica). Tricine SDS-PAGE indicated that 5 k- and 6.5 k-AGRPs are single polypeptides, but 12.5 k- and 14 k-AGRPs consist of two polypeptide chains, which are linked by disulfide bond(s). The N-terminal amino acid sequences of four AGRPs were analyzed by a gas-phase sequencer, and the result indicated that they are distinct molecules. Comparison of the sequences with those of proteins in the protein data base demonstrates that 5 k- and 6.5 k-AGRPs have a significant homology with a basic peptide from pumpkin seeds and with cocoa seed vicilin, respectively, and that 12.5 k- and 14 k-AGRPs are related to 2S seed storage proteins. Furthermore, it was assumed that the four AGRPs might occur in the protein bodies within cells of the seed.

• Murphy ME, Lindley PF, Adman ET
• Structural comparison of cupredoxin domains: domain recycling to construct proteins with novel functions.
• Protein Sci. 1997; 6: 761-70
• Display abstract

• The three-dimensional structures of the copper-containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of the enzyme domains and their known cupredoxin relatives shows clearly that there are at least six cupredoxin classes, and that the evolution of the conserved core of these domains is independent of the presence or absence of copper sites. Relationships among the variable loops in these structures show that the two-domain ancestor of the blue oxidases contained a trinuclear-copper interface but could not have functioned in a monomeric state. Comparison of the sequence of the copper-containing, iron-regulating protein. Ferrous transport (Fet3) from yeast to the structurally defined core and loop residues of the cupredoxins suggests specific residues that could be involved in the ferroxidase activity of Fet3.

• Odaira M, Yoshida S, Maeshima M
• Accumulation of a glycoprotein that is homologous to a seed storage protein in mung bean hypocotyls at the late stage of tissue elongation.
• Plant Cell Physiol. 1997; 38: 290-6
• Display abstract

• Physiological changes were examined in the amount of a 50-kDa glycoprotein (gp50) that was recovered in a nuclear fraction from hypocotyls of mung bean (Vigna radiata) seedlings. Immunoblot analysis indicated that the glycoprotein was present in hypocotyls and epicotyls from 4- and 5-day-old seedlings but not in hypocotyls from 2-day-old seedlings. The glycoprotein was not detected in leaves or roots. When we divided hypocotyls of 3-day-old seedlings into elongating region (0 to 1.5 cm below the cotyledon) and the mature region, we found gp50 in the mature region only. The results suggest that the 50-kDa glycoprotein is synthesized de novo and accumulates at the late stage during elongation of cells in the hypocotyl. Furthermore, an antibody specific to gp50 reacted with a major 50-kDa protein in cotyledons, which is known as a storage protein in mung bean cotyledon. Eighteen amino acid residues among 22 amino-terminal residues of gp50 were identical to those of the storage protein from cotyledon. A peptide map of the glycoprotein after digestion with V8 protease was similar to that of the storage protein. Overall, our findings suggest that the glycoprotein recovered in the nuclear fraction is an isoform of the seed storage protein that is expressed only in the mature cells of hypocotyls and epicotyls.

• Baumlein H, Braun H, Kakhovskaya IA, Shutov AD
• Seed storage proteins of spermatophytes share a common ancestor with desiccation proteins of fungi.
• J Mol Evol. 1995; 41: 1070-5
• Display abstract

• The legumin- and vicilin-like seed storage globulins of spermatophytes are specifically accumulated during embryogenesis and seed development. Previous studies have shown that a precursor common to both legumin and vicilin genes might have evolved by duplication from a single-domain ancestral gene. We here report that amino acid sequences of legumin and vicilin domains share statistically significant similarity to the germination-specific germins of wheat as well as to the spherulation-specific spherulins of myxomycetes. This conclusion is further supported by the derived intron-exon structure of a spherulin gene. Spherulins are thought to be involved in tissue desiccation or hydration. It is suggested that the present-day seed globulins of spermatophytes have evolved from a group of ancient proteins functional in cellular desiccation/hydration processes.

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