School of Plant Sciences, University of Reading, Reading, England
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Abstract |
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Introduction |
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The cupin superfamily was discovered when amino acid sequence analysis, originally based on the nonapeptide "germin-box" (Lane et al. 1991
), found a high level of similarity between fungal spherulins, produced upon spore formation in the slime mold Physarum polycephalum, and wheat germin, a thermostable, glycosylated protein produced during germination and shown to have oxalate oxidase (OXO) (EC 1.2.3.4) activity (Lane et al. 1993
; Lane 2000
). This small family of germin-related proteins was then found to share a small number of globally conserved residues with 7S (vicilin) and 11S (legumin) seed storage proteins (Bäumlein et al. 1995
). Subsequent analysis (Dunwell and Gane 1998
), using an extended version of the germin-box, identified the conserved domain in a large number of microbial proteins. These included type II phosphomannose isomerases (PMIs) (EC 5.3.1.8), found in a large number of prokaryotes, AraC-type (Gallegos et al. 1997
) and other (Aravind and Koonin 1999b
) transcription regulators, gentisate 1,2-dioxygenases (GDOs) (EC 1.13.11.4), and oxalate decarboxylases (OXDCs) (EC 4.1.1.2).
Although some functional information is available, the majority of bacterial and archaeal cupins, as well as an increasing number from plants (generally known as germin-like proteins [GLPs]) and animals, have been identified from genome sequencing projects and are of unknown function. Together with the experimental confirmation of the bifunctional nature of barley germin as an enzyme with both OXO and manganese superoxide dismutase (Mn-SOD) (EC 1.15.1.1) activity (Woo et al. 2000
), as well as the recent evidence that a GLP from moss (Yamahara et al. 1999
) and one from tobacco (Carter and Thornburg 2000
) are Mn-SODs, there is circumstantial evidence relating to the possible functions of many other germins and GLPs. Some are expressed at critical developmental stages such as embryogenesis (Domon et al. 1995
; Neutelings 1998
) or floral induction (Heintzen et al. 1994
; Staiger, Apel, and Trepp 1999
), and many are induced by a range of stresses, either biotic, such as infection with powdery mildew (Thordahl- Christensen et al. 1997
; Schweizer, Christoffel, and Dudler 1999
), or abiotic, such as exposure to salt (Hurkman and Tanaka 1996
), aluminum (Hamel, Breton, and Houde 1998
), or high temperatures (Vallelian-Bindschedler et al. 1998
).
Cupins have two histidine-containing motifs (fig. 1
), corresponding to the C/D and G/H strands of OXO (Woo et al. 2000
), separated by an intermotif region (IMR) which includes strands E and F and the intervening loop. The IMR of the single domain cupins varies in length from 15 amino acids in most archaeal and bacterial cupins to 26 residues in some of the cereal proteins (Dunwell, Khuri, and Gane 2000
). It is presumed that this variation in intermotif length, due principally to insertions in the interstrand loop, delayed the identification of the conserved residues in this diverse superfamily of proteins. It is now known that the two conserved histidines and a glutamate in the first motif, together with a third conserved histidine in the second motif (fig. 1
), act as ligands for the active-site metal, which has been shown to be a single manganese atom for barley germin (Requena and Bornemann 1999
; Woo et al. 2000
), and for the GLPs from a moss (Barbula unguiculata; Yamahara et al. 1999
) and from tobacco (Carter and Thornburg 2000
).
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Materials and Methods |
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The various sequences were identified using BLAST searches with a variety of cupin sequences from different species. The nonredundant, nonmouse, and nonhuman EST and microbial GenBank databases at the National Institute for Biotechnology Information (NIH, Bethesda, Md.) were mainly used via http://www.ncbi.nlm.nih.gov/BLAST; other sequences were identified by using the Institute of Genome Research TIGR database (Rockville, Md.) at http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi and the Sanger Centre database (Cambridge, England) at http://sanger.ac.uk. To avoid errors and misleading results, all microbial sequences used in these analyses were from completed genomes, except for the Streptococcus mutans bi-cupin putative OXDC. Similarly, identical cupin sequences (e.g., from Arabidopsis thaliana) were deleted, and only a small number of plant ESTs were used in the phylogenetic studies. General alignments were performed using CLUSTAL W at the BCM Search Launcher at http://dot.imgenn.bcm.tmc.edu:9331/ and manually adjusted to produce a largely unambiguous alignment. Subsequent phylogenetic analysis was performed using PAUP*, version 4.0b4a (Swofford 1999
). A global alignment was constructed with a total of 120 protein sequences comprising 25 plant GLPs, 5 fungal sequences, and 90 microbial sequences, of which 13 were from archaeal species. A second separate alignment was constructed, containing 73 GLPs from plants and fungi. Results of the unrooted global analysis indicated that ePBA was an appropriate root for the GLP analysis (see below).
As described above, phylogenetic analyses were performed for both alignments with the IMR region included; excluding this region from the analyses resulted in loss of resolution in the topologies found. Parsimony searches in both analyses involved heuristic searches with the following settings: gaps were treated as "missing"; branches were collapsed if maximum branch length was zero; topological constraints were not enforced, and STEEPEST DESCENT, ACCTRAN, and MULPARS were in effect. In order to measure clade support, jackknife analysis was carried out using PAUP with settings emulating Parsimony Jackknifer (Farris 1969
), i.e., percentage of characters deleted in each replicate = 37, "fast" stepwise addition, "Jac" resampling, and only groups with jac frequency >50% kept. Initial (equally weighted) searches were constrained by jackknife trees thus obtained so as to avoid searching through irrelevant parts of tree space. Given the expectation that the sequences in our data sets could be highly divergent, the "Protpars" stepmatrix as implemented in MacClade (version 3.0.8a; Maddison and Maddison 1992
) was used in subsequent searches in order to take into account differences in probabilities of substitutions among amino acids (i.e., stepmatrix values are based on the minimum number of changes between amino acids, calculated from the genetic code).
The 120-protein sequence alignment contained 70 positions, of which 25 were part of the IMR. In total, the alignment contained 65 variable positions, of which 62 were phylogenetically informative. A heuristic parsimony search was performed with 100 random sequence additions ("hold" = 3) without branch swapping, with "MULPARS off," and saving trees even when they were not optimal overall. The resulting trees were then used as starting trees in a search with "MULPARS on" and TBR branch swapping, which yielded >20,300 most-parsimonious trees (MPTs) of length 2,039 (consistency index [CI] = 0.35, retention index [RI] = 0.57). The search was repeated with the Protpars stepmatrix implemented in order to reflect generally observed substitution patterns in amino acid sequences. This yielded 6 MPTs of length 2,688 (CI = 0.26, RI = 0.59), of which the strict consensus, with corresponding jackknife support values, was taken as the final result. This stepmatrix-weighted strict consensus tree was largely congruent with and better resolved than the unweighted strict consensus (not shown).
The sequence alignment of 73 plant GLPs contained 64 positions, of which 53 were phylogenetically informative. A heuristic parsimony search with 50 replicates of random sequence addition (holding three trees per step), with TBR branch swapping, and keeping only trees compatible with a jackknife tree calculated from the same data set yielded 3,660 MPTs of length 631 (CI = 0.49, RI = 0.75). Repeating the analysis with the Protpars stepmatrix implemented and without topological constraints yielded 2,928 MPTs of weighted length 761 (CI = 0.42, RI = 0.76), of which the strict consensus tree, rooted with the "ePBA" clade, is presented below. This rooting was based on the global topology (see above). As with the global analysis, the effect of implementing the Protmars stepmatrix was a further increase in resolution in the plant-weighted strict consensus topology compared with that from the unweighted analysis (not shown).
Each sequence used in this study is denoted by a three-letter code made up of the first letter of the generic name and the first two letters of the epithet, with prokaryotic abbreviations in lowercase and eukaryotic ones in uppercase; e.g., "pae" denotes Pseudomonas aeruginosa, and "HVU" denotes Hordeum vulgare. Tables 1 and 2 contain a complete listing of the sequences used.
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Results and Discussion |
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The other sequences used were of unknown function. Several changes in IMR length have occurred across the phylogeny (fig. 2 ), from the basic, ancestral value of 15 residues found in the PMIs and transcriptional regulators to a maximum of 26 in a small number of cereal GLPs (see below).
The PMI Cluster
Within the monophyletic PMI clade (fig. 2
), there are two key observations to note. First, the A. aeolicus (gi|2983213, aae) sequence is on a relatively long branch derived from within the PMI group, a pattern consistent with hypotheses about the ancient origin of this bacterium (Deckert et al. 1998
). This cupin has only two conserved histidines; the glutamate and third histidine that complete the putative active site are substituted by valine and tyrosine, respectively. There is only a single nucleotide difference between glutamate (GAA/G) and valine (GTN) codons and between those encoding histidine (CAC/T) and tyrosine (TAC/T).
Second, the position of the cyanobacterial Anacystis nidulans (gi|1405429, ani) sequence as sister to all the other PMIs is intriguing. The likely function of this sequence as a PMI was verified by its similarity (e value 0.001) to a P. aeruginosa PMI (gi|3510759, pae5), although its most similar neighbors were two archaeal sequences from Pyrococcus horikoshii (gi|3257338, pho3, 4e-5) and Pyrococcus abyssi (gi|5458652, pab, 8e-5). In fact, all the archaeal PMI-like sequences group together after "ani," forming a sister clade to the rest of the PMIs. This placement of the archaeal PMI-like sequences between a gram-negative bacterium and the gram-positive clades concurs with theories on the chimeric origin of the archaeal genome (Koonin et al. 1997
).
Other Functional Groupings
The OXDC bi-cupins that were used for this analysis formed one clearly identified monophyletic clade. They included two fungal proteins: fCVE (gi|6468006) from Collybia velutipes (now known as Flammulina velutipes) (Kesarwani et al. 2000
), and fAPH from Aspergillus phoenices (sequence from Scelonge and Bidney [1998
], as amended by Dunwell, Khuri, and Gane [2000]
), along with putative OXDC sequences from Bacillus subtilis (gi|2635837, bsu6; gi|2619026, bsu7), Synechocystis (gi|1652630, syn5) (Dunwell 1998b
), and S. mutans (Contig205, DNA positions 69787153 and 75187688, smu). These analyses were conducted before recent experimental evidence confirmed bsu6 to be an OXDC (Tanner and Bornemann 2000
). This finding, coupled with the high level of sequence similarity between these proteins, lends further support to the prediction that bsu7, syn5, and smu are OXDCs.
Although the two cupin domains (A and B) of each OXDC were treated separately in the analysis, all OXDC sequences formed one clade within which the two domains clustered as sister groups (fig. 2 ). The most likely interpretation of this is that a duplication event occurred once in the lineage leading to the OXDC clade, followed by divergence of the sequences. However, further analysis is needed before a conclusive statement is made regarding the evolution of the bi-cupins.
The only other group of proteins with known function that did not group consistently together in our analyses were the transcription regulators (TRs) (fig. 2
). Most of those used in this study belong to the bacterial AraC/XylS family delineated by Gallegos et al. (1997)
on the basis of the high sequence similarity of their helix-turn-helix DNA-binding domains; the cupin motifs occur within the N-terminal effector-binding domains of these proteins. This arrangement contrasts with that in the other TRs from thermophilic archaea and bacteria (gi|2621786, mth1; gi|2621742, mth2; gi|4981179, tmar1), in which the cupin domain is located at the C- terminal end of the protein (Aravind and Koonin 1999b
). Whereas these three sequences clustered closer together, most of the AraC-type sequences were more dispersed. Two rhamnose-binding TRs, namely, Escherichia coli rhaS (gi|305009, eco5) and Salmonella typhimurium rhaC2 (gi|47906, sty), did form a group, and a third rhamnose-binding TR, rhaR (gi|305010, eco4) from E. coli, was nearby. Similarly, an E. coli chitobiose-binding TR (gi|1742832, eco3) (formerly considered to bind cellobiose), grouped with a B. subtilis TR (gi|2632741, bsu2). It is therefore possible that this dispersal might be a result of the range of effectors (sugars or other compounds) involved in binding to the cupin domain.
The alignment provided in figure 3
shows additional evidence of the similarity between the TRs and highlights three points. First, the AraC-type group can be distinguished clearly from the thermophilic group in having only one of the conserved histidine residues in motif 1. Second, the archetypal arabinose-binding E. coli AraC (gi|40936, eco2), although similar to other cupins in terms of its beta-barrel structure (Soisson et al. 1997
; Dunwell, Khuri, and Gane 2000
), is not typical of its group in terms of sequence. Third, the two sequences from B. subtilis (gi|1881323, bsu1) and Mycobacterium tuberculosis (gi|2213518, mtu2) both have a glutamine residue in place of the glutamate in the putative binding site of these proteins. A more comprehensive alignment of 112 TRs and putative TR sequences showed that a total of 54 proteins with a glutamine at that position compared with 33 that had a glutamate residue (data not shown). This contrasts with the higher conservation frequencies of the three histidines, which were 94, 95, and 104, respectively.
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Plant and Fungal Germin-like Proteins
Phylogenetic Analysis
The plant GLPs used in this study included examples from most main plant lineages, including cereals, legumes, gymnosperms, and two moss GLPs (B. unguiculata, gi|6429233, mBUN; Ceratodon purpureus EST gi|6068694, emCPU). In addition, two fungal spherulins from P. polycephalum (gi|161262 and gi|134860; fPPO1 and 2) were included. The resulting phylogeny contained three main groups (fig. 4
), a result which differs from that recently proposed by Carter and Thornburg (1999, 2000)
, who identified five separate clades. However, Carter and Thornburg produced their phylogeny using the whole protein sequence, rather than only the cupin domain. Furthermore, although the total number of sequences used for the phylogenies was coincidentally the same, Carter and Thornburg used 30 sequences from A. thaliana, 10 from Oryza sativa and 33 GLPs from other plant species, including one moss, whereas this analysis utilized 24 from A. thaliana, 11 from O. sativa, and 36 from other plant species, including (see above) two moss species and two from a fungus (table 2
). The phylogeny presented in figure 4
, therefore, gives a more complete picture of the evolutionary, as well as the functional, relationships among GLPs.
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Subfamily 2 (s/f 2) includes GLPs from a wide range of taxonomic groups, including other cereals, gymnosperms, and halophytic species, namely Atriplex lentiformis (gi|4996622, ALE) and Mesembryanthemum crystallinum (gi|167258, MCR). Furthermore, these cupins were isolated from diverse and seemingly unconnected sources. The solanaceous group, for example, consisted of GLPs from Mn-deficient roots of tomato (gi|2979494, LES), nectar of tobacco (gi|6090828, NPL), and cell suspension cultures of potato (gi|3171251, STU). The two gymnosperm GLPs were both isolated from somatic embryos (Neutelings 1998
).
Despite this diversity of origin, nested within this subfamily is a group of proteins linked directly to stress tolerance: MCR was isolated from salt-stressed tissue, and OSA2 (gi|5499730) and OSA5 (gi|5499732) were labeled stress-responsive root GLPs. In addition, two centrally positioned proteins within s/f 2 are now known to have Mn-SOD activity, namely NPL (gi|6090828) from Nicotiana plumbaginifolia (Carter and Thornburg 2000
) and mBUN (gi|6429233) from the moss B. unguiculata (Yamahara et al. 1999
). It is therefore possible to predict that the GLPs in s/f 2 all have a role in tolerance to oxidative stress and are most likely to be Mn- SODs. This is in accordance with the predictions of Carter and Thornburg (2000)
, and it is expected that more experimental evidence will be forthcoming which will confirm these suggestions.
The third grouping, subfamily 3 (s/f 3), includes low-affinity auxin-binding proteins from peach (gi|4098517, PPE1; gi|1916809, PPE2; gi|1916807, PPE3), as well as GLPs known to have a role linked to circadian rhythms and floral induction in A. thaliana (gi|1755184, ATH25; Staiger, Apel, and Trepp 1999
), Sinapis alba (gi|683488, SAL; Heintzen et al. 1994
), and Pharbitis nil (gi|662292, PNI; Ono et al. 1996
). This subfamily may therefore consist mainly of regulatory proteins involved with auxin metabolism, either directly or indirectly.
The Metal-Binding Active Site
As seen above, with few exceptions, the enzymatic function(s) of GLPs remain largely unknown. It is clear that most of them contain a single metal-binding active site with three conserved histidine ligands and one glutamate ligand (fig. 1
). The position of the active site in the protein is protected within the beta-barrel structure (Woo et al. 2000
), and it has recently been shown that the barley germin with OXO and SOD activity (Requena and Bornemann 1999
; Woo et al. 2000
), and the moss (Yamahara et al. 1999
) and tobacco (Carter and Thornbug 2000
) GLPs with SOD activity are manganese-containing enzymes. Both of these enzymes generate hydrogen peroxide, OXO by the oxidation of the oxalate ion, and SOD by the dismutation of superoxide radicals. It is likely that the types of catalysis are similar in all metal-binding GLPs or that there is a group, possibly s/ f 3 as delineated above, that contains a different metal and therefore has slightly different properties.
Interestingly, a small number of GLPs do not contain all four of the highly conserved active-site residues. Instead of the third histidine (coded by CAY) residue in the second motif, PNI (gi|662292) has an aspartate (GAY), while ATH4 (gi|6721170), ATH5 (gi|6721168), and ATH7 (gi|6721166) have a glutamine (CAR). The lack of the third histidine infers that these proteins may not bind a manganese atom, or that they bind it very loosely and only under certain pH conditions. The conserved glutamate (GAR) in the first cupin motif can be substituted, again, by either aspartate (GAY), as in TAE8 (gi|1772601), or glutamine (CAR), as in mCPU (gi|6068694), ATH6 (gi|6721167), and ATH4 (gi|6721170). The absence of the negatively charged glutamate makes it unlikely that these proteins would bind manganese, and it is not known whether the equally negatively charged aspartate would provide the same function. In some Glycine max ESTs (gi|5759981, gi|6725711, and gi|6566187), this position is occupied by a positively charged lysine (AAR). Since all of these changes are due to a single nucleotide difference, they may simply be mutations that maintain the structure of these proteins but alter the binding characteristics of the active site and thus change its function.
The Intermotif Region
In the plant and fungal single-domain GLPs examined to date, the intermotif region varies in length from the usual minimum of 20 (exceptionally, 19 in some legume sequences) to 26 amino acids in some barley and wheat sequences (fig. 4
). There is a high level of conservation within the IMR across all of the cupin subfamilies identified above, at both the protein and the DNA levels. An alignment of the DNA sequences (fig. 5
) encoding the GLPs used in the global analysis allows the identification of certain patterns within the region encoding the IMR. First, two GLPs (PPE1 gi|4098517 and PSA1 gi|2739260) show evidence of two compensatory frameshifts. Second, where the IMR is longer than 20 amino acids, the position of the additional residues is consistently within the loop between structural strands E and F, to either side of a conserved asparagine. Third, the alignment shows a 100% conservation of thymine in the second base positions of three codons within strand E and two codons in strand F. Apart from methionine, codons with thymine in the second position code for leucine (L), isoleucine (I), valine (V), and phenylalanine (F), all of which are hydrophobic residues (Trinquier and Sanejouand 1998
) and may help to maintain the intermonomer interactions necessary for the assembly of the homohexamers required for enzyme activity in germins (see references in Dunwell, Khuri, and Gane 2000
). These observations are also linked to conservation of the core region of the barrel structure of cupins, in that the length and composition of the ß-strands remains largely conserved, with most variability occurring in the structurally less important central loop region between strands E and F.
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Conclusions |
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An unrooted phylogeny of all the known cupin motifs at the time of analysis (tables 1 and 2
) gave rise to a function-based clustering (fig. 2
), proving that even within the six-strand cupin domain there was sufficient information to permit discrimination between different clades. If this tree were to be rooted using the cupin (aae) from the thermophilic A. aeolicus (considered the most deeply branched bacterium; Deckert et al. 1998
) as the outgroup, it is expected that bacterial PMIs would be the most primitive cupins, with the eukaryotic GLPs, and particularly the true germins (from cereals), being the most advanced.
In terms of functional evolution, this means that the first "protocupin" arose as a small ß-barrel protein (about 100 residues), perhaps containing iron or manganese (Kirschvink et al. 2000
) and initially able to bind a variety of simple sugars. As the environment changed, the protocupin gene was modified and diversified as the prokaryote evolved. The proteins underwent amino acid additions and/or substitutions to bind more complex sugars and fused with other "protoproteins" to catalyze more complex reactions. Throughout this evolutionary process, the key structural residues were conserved and the distinctive beta-barrel structure was maintained. At some point, one such cupin fused to another, or was duplicated, with the emergence of bi-cupins such as the OXDCs. It is hoped that the data set from this superfamily of proteins will be of value in addressing the objections raised against phylogenetic analyses by those who argue that most sequences are heavily saturated with respect to amino acid substitutions and that it is usually not possible to define a stable character in a given domain (a specific conserved residue) because these positions have evolved too rapidly (Philippe and Forterre 1999
). In the cupin sequences, in contrast, there are definite fixed residues known to have important structural and functional significance.
Relationship of GLPs to other Mn-SODs
Recently, two independent sources have provided evidence for a functional similarity between GLPs and the well-characterized group of iron/manganese SODs found in prokaryotes and in the mitochondria of eukaryotes. First, resolution of the three-dimensional structure of barley germin at 1.6 Å (Woo et al. 2000
) revealed an active site comprising a single Mn atom liganded by the three histidines and the single glutamate within the two cupin motifs (fig. 1
). This active site has close geometric similarity to that of the Mn/Fe class of SOD, which consists of three histidines and an aspartate residue (although the overall protein fold of these SODs is different from that of the cupins). Direct experimental evidence for the connection comes from the finding of SOD activity in a manganese-containing GLP from the moss B. unguiculata (Yamahara et al. 1999
), from the detection of Mn-SOD activity from a tobacco GLP (Carter and Thornburg 2000
), and from the detection of both OXO and SOD activities in a barley germin (Woo et al. 2000
).
It now seems logical to suggest that the major functional role for all GLPs is as a SOD enzyme that is active at neutral pH and protects plants from oxidative stress induced by the range of biotic and abiotic stresses referred to above. Compared with the majority of plant GLPs, the more specialized cereal germins (figs. 2 and 5
) are assumed to have evolved an additional OXO function at low pH. This activity provides the additional benefit of enabling detoxification of the oxalic acid produced by several plant pathogens (Dunwell, Khuri, and Gane 2000
).
It is now thought that the ancestral form of the previously characterized type of microbial SOD was cambialistic (i.e., it was able to bind both Fe and Mn) and that the two isoforms (Mn form and Fe form) diverged at a later stage of evolution, after the divergence of the Archaea and Eubacteria. This change in metal binding specificity has recently been related to changes in the redox potential of the surface oceans during the periods around the intense global glaciations known as "snowball earth" (Kirschvink et al. 2000
). The enzymatic cupin precursors may have undergone a similar evolutionary path.
As this study has shown, in this genome-sequencing era, it is possible to continue to define new structurally related families and superfamilies of functionally diverse proteins. It is imperative, however, that predictions of the enzymatic functions of proteins be supported by biochemical evidence, although this is not always a simple issue. The identification of barley germin as an OXO (Lane et al. 1993
), for example, came 80 years after evidence for the enzyme was first provided (Zaleski and Reinhard 1912
), and it was nearly 10 years later that its second function as a SOD was discovered (Woo et al. 2000
).
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Acknowledgements |
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Footnotes |
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1 Present address: Wageningen University, Plant Taxonomy Group, Wageningen, the Netherlands
2 Keywords: phylogeny
cupin
germin
oxalate oxidase
phosphomannose isomerase
protein structure
3 Address for correspondence and reprints: Jim M. Dunwell, School of Plant Sciences, University of Reading, Reading RG6 6AS, United Kingdom. j.m.dunwell{at}reading.ac.uk
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