NORMAL GENOMIC

• Nakata M et al.
• Germin-like protein gene family of a moss, Physcomitrella patens,phylogenetically falls into two characteristic new clades.
• Plant Mol Biol. 2004; 56: 381-95
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• We identified 77 EST clones encoding germin-like proteins (GLPs) from amoss, Physcomitrella patens in a database search. These PhyscomitrellaGLPs ( PpGLP s) were separated into seven groups based on DNA sequencehomology. Phylogenetic analysis showed that these groups were divided intotwo novel clades clearly distinguishable from higher plant germins andGLPs, named bryophyte subfamilies 1 and 2. PpGLPs belonging to bryophytesubfamilies 1 lacked two cysteines at the conserved positions observed inhigher plant germins or GLPs. PpGLPs belonging to bryophyte subfamily 2contained two cysteines as observed in higher plant germins and GLPs. Inbryophyte subfamily 1, 12 amino acids, in which one of two cysteines isincluded, were deleted between boxes A and B. Further, we determined thegenomic structure of all of seven PpGLP genes. The sequences of PpGLP s ofbryophyte subfamily 1 contained one or two introns, whereas those ofbryophyte subfamily 2 contained no introns. Other GLPs from bryophytes, aliverwort GLP from Marchantia polymorpha , and two moss GLPs from Barbulaunguiculata and Ceratodon purpureus also fell into bryophyte subfamily 1and bryophyte subfamily 2, respectively. No higher plant germins and GLPswere grouped into the bryophyte subfamilies 1 and 2 by our analysis.Moreover, we revealed that PpGLP6 had manganese-containing extracellularsuperoxide dismutase activity. These results indicated that bryophytepossess characteristic GLPs, which phylogenetically are clearlydistinguishable from higher plant GLPs.

• Dunwell JM, Purvis A, Khuri S
• Cupins: the most functionally diverse protein superfamily?
• Phytochemistry. 2004; 65: 7-17
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• The cupin superfamily of proteins, named on the basis of a conservedbeta-barrel fold ('cupa' is the Latin term for a small barrel), wasoriginally discovered using a conserved motif found within germin andgermin-like proteins from higher plants. Previous analysis of cupins hadidentified some 18 different functional classes that range fromsingle-domain bacterial enzymes such as isomerases and epimerases involvedin the modification of cell wall carbohydrates, through to two-domainbicupins such as the desiccation-tolerant seed storage globulins, andmultidomain transcription factors including one linked to the nodulationresponse in legumes. Recent advances in comparative genomics, and theresolution of many more 3-D structures have now revealed that the largestsubset of the cupin superfamily is the 2-oxyglutarate-Fe(2+) dependentdioxygenases. The substrates for this subclass of enzyme are many andvaried and in total amount to probably 50-100 different biochemicalreactions, including several involved in plant growth and development.Although the majority of enzymatic cupins contain iron as an active sitemetal, other members contain either copper, zinc, cobalt, nickel ormanganese ions as a cofactor, with each cofactor allowing a different typeof chemistry to occur within the conserved tertiary structure. This reviewdiscusses the range of structures and functions found in this most diverseof superfamilies.

• Khuri S, Bakker FT, Dunwell JM
• Phylogeny, function, and evolution of the cupins, a structurallyconserved, functionally diverse superfamily of proteins.
• Mol Biol Evol. 2001; 18: 593-605
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• The cupin superfamily is a group of functionally diverse proteins that arefound in all three kingdoms of life, Archaea, Eubacteria, and Eukaryota.These proteins have a characteristic signature domain comprising twohistidine- containing motifs separated by an intermotif region of variablelength. This domain consists of six beta strands within a conserved betabarrel structure. Most cupins, such as microbial phosphomannose isomerases(PMIs), AraC- type transcriptional regulators, and cereal oxalate oxidases(OXOs), contain only a single domain, whereas others, such as seed storageproteins and oxalate decarboxylases (OXDCs), are bi-cupins with two pairsof motifs. Although some cupins have known functions and have beencharacterized at the biochemical level, the majority are known only fromgene cloning or sequencing projects. In this study, phylogenetic analyseswere conducted on the conserved domain to investigate the evolution andstructure/function relationships of cupins, with an emphasis on single-domain plant germin-like proteins (GLPs). An unrooted phylogeny of cupinsfrom a wide spectrum of evolutionary lineages identified three mainclusters, microbial PMIs, OXDCs, and plant GLPs. The sister group to theplant GLPs in the global analysis was then used to root a phylogeny of allavailable plant GLPs. The resulting phylogeny contained three main clades,classifying the GLPs into distinct subfamilies. It is suggested that thesesubfamilies correlate with functional categories, one of which containsthe bifunctional barley germin that has both OXO and superoxide dismutase(SOD) activity. It is proposed that GLPs function primarily as SODs,enzymes that protect plants from the effects of oxidative stress. Closerinspection of the DNA sequence encoding the intermotif region in plantGLPs showed global conservation of thymine in the second codon position, acharacter associated with hydrophobic residues. Since many of theseproteins are multimeric and enzymatically inactive in their monomericstate, this conservation of hydrophobicity is thought to be associatedwith the need to maintain the various monomer- monomer interactions. Thetype of structure-based predictive analysis presented in this paper is animportant approach for understanding gene function and evolution in an erawhen genomes from a wide range of organisms are being sequenced at a rapidrate.

• Chen Z
• A superfamily of proteins with novel cysteine-rich repeats.
• Plant Physiol. 2001; 126: 473-6

• Dunwell JM, Khuri S, Gane PJ
• Microbial relatives of the seed storage proteins of higher plants:conservation of structure and diversification of function during evolutionof the cupin superfamily.
• Microbiol Mol Biol Rev. 2000; 64: 153-79
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• This review summarizes the recent discovery of the cupin superfamily (fromthe Latin term "cupa," a small barrel) of functionally diverse proteinsthat initially were limited to several higher plant proteins such as seedstorage proteins, germin (an oxalate oxidase), germin-like proteins, andauxin-binding protein. Knowledge of the three-dimensional structure of twovicilins, seed proteins with a characteristic beta-barrel core, led to theidentification of a small number of conserved residues and thence to thediscovery of several microbial proteins which share these key amino acids.In particular, there is a highly conserved pattern of twohistidine-containing motifs with a varied intermotif spacing. This cupinsignature is found as a central component of many microbial proteinsincluding certain types of phosphomannose isomerase, polyketide synthase,epimerase, and dioxygenase. In addition, the signature has been identifiedwithin the N-terminal effector domain in a subgroup of bacterial AraCtranscription factors. As well as these single-domain cupins, this surveyhas identified other classes of two-domain bicupins including bacterialgentisate 1, 2-dioxygenases and 1-hydroxy-2-naphthoate dioxygenases,fungal oxalate decarboxylases, and legume sucrose-binding proteins. Cupinevolution is discussed from the perspective of the structure-functionrelationships, using data from the genomes of several prokaryotes,especially Bacillus subtilis. Many of these functions involve aspects ofsugar metabolism and cell wall synthesis and are concerned with responsesto abiotic stress such as heat, desiccation, or starvation. Particularemphasis is also given to the oxalate-degrading enzymes from microbes,their biological significance, and their value in a range of medical andother applications.

• Sachetto-Martins G, Franco LO, de Oliveira DE
• Plant glycine-rich proteins: a family or just proteins with a commonmotif?
• Biochim Biophys Acta. 2000; 1492: 1-14
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• Twelve years ago a set of glycine-rich proteins (GRP) of plants werecharacterized and since then a wealth of new GRPs have been identified.The highly specific but diverse expression pattern of grp genes, takentogether with the distinct sub-cellular localisation of some GRP groups,clearly indicate that these proteins are implicated in several independentphysiological processes. Notwithstanding the absence of a clear definitionof the role of GRPs in plant cells, studies conducted with these proteinshave provided new and interesting insights on the molecular and cellbiology of plants. Complex regulated promoters and distinct mechanisms ofgene expression regulation have been demonstrated. New protein targetingpathways, as well as the exportation of GRPs from different cell typeshave been discovered. These data show that GRPs can be useful as markersand/or models to understand distinct aspects of plant biology. In thisreview, the structural and functional features of this family of plantproteins will be summarised. Special emphasis will be given to the geneexpression regulation of GRPs isolated from different plant species, as itcan help to unravel their possible biological functions.

• Meyer K, Keil M, Naldrett MJ
• A leucine-rich repeat protein of carrot that exhibits antifreeze activity.
• FEBS Lett. 1999; 447: 171-8
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• A gene encoding an antifreeze protein (AFP) was isolated from carrot(Daucus carota) using sequence information derived from the purifiedprotein. The carrot AFP is highly similar to the polygalacturonaseinhibitor protein (PGIP) family of apoplastic plant leucine-rich repeat(LRR) proteins. Expression of the AFP gene is rapidly induced by lowtemperatures. Furthermore, expression of the AFP gene in transgenicArabidopsis thaliana plants leads to an accumulation of antifreezeactivity. Our findings suggest that a new type of plant antifreeze proteinhas recently evolved from PGIPs.

• Shutov AD, Braun H, Chesnokov YV, Baumlein H
• A gene encoding a vicilin-like protein is specifically expressed in fernspores. Evolutionary pathway of seed storage globulins.
• Eur J Biochem. 1998; 252: 79-89
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• The isolation and characterisation of a cDNA coding for a vicilin-likeprotein of the fern Matteuccia struthiopteris is described. Thecorresponding gene is specifically expressed during late stages of sporedevelopment. Extensive sequence comparisons suggest that the fern proteincan be considered as a molecular missing link between single-domaingermin/spherulin-like proteins and two-domain seed storage globulins ofgymnosperms and angiosperms. Further, evidence is provided for theexistence of a superfamily of structurally related, functionally differentproteins which includes storage globulins of the vicilin and leguminfamilies, a membrane-associated sucrose-binding protein of soybean, aForssman antigen-binding lectin of velvet bean, the precursor of thevacuolar membrane bound proteins MP27/MP32 of pumpkin, theembryogenesis-specific protein Gea8 of carrot, the fern-spore-specificprotein described here as well as the functionally diverse family ofgermins/germin-like proteins and the spherulins of myxomycetes. We proposethat seed storage globulins of spermatophytes evolved fromdesiccation-related single-domain proteins of prokaryotes via a duplicatedtwo-domain ancestor that is best represented by the extant fernspore-specific vicilin-like protein.

• Gane PJ, Dunwell JM, Warwicker J
• Modeling based on the structure of vicilins predicts a histidine clusterin the active site of oxalate oxidase.
• J Mol Evol. 1998; 46: 488-93
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• It is known that germin, which is a marker of the onset of growth ingerminating wheat, is an oxalate oxidase, and also that germins possesssequence similarity with legumin and vicilin seed storage proteins. Thesetwo pieces of information have been combined in order to generate a 3Dmodel of germin based on the structure of vicilin and to examine the modelwith regard to a potential oxalate oxidase active site. A cluster of threehistidine residues has been located within the conserved beta-barrelstructure. While there is a relatively low level of overall sequencesimilarity between the model and the vicilin structures, the conservationof amino acids important in maintaining the scaffold of the beta-barrellends confidence to the juxtaposition of the histidine residues. Thecluster is similar structurally to those found in copper amine oxidase andother proteins, leading to the suggestion that it defines a metal-bindinglocation within the oxalate oxidase active site. It is also proposed thatthe structural elements involved in intermolecular interactions invicilins may play a role in oligomer formation in germin/oxalate oxidase.

• Tsyguelnaia I, Doolittle RF
• Presence of a fibronectin type III domain in a plant protein.
• J Mol Evol. 1998; 46: 612-4
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• A hidden Markov model (HMM) approach was used to identify potential candidates in sequence databases for fibronectin type III domains in plants, a kingdom heretofore bereft of these structures. Fortuitously, one of the proteins uncovered had already had a crystal structure published, allowing direct structural confirmation of the existence of this domain in plants.