BACKGROUND
plants are continuously challenged by pathogens, herbivores and adverse environmental conditions.
although they lack an adaptive immune system, they have evolved numerous other mechanisms of stress response and defence.
these include developmental plasticity, mechanical barriers, low molecular weight anti-microbial compounds named phytoalexins, pathogen-specific resistance genes and inducible pathogenesis-related  proteins.
pr-proteins were first discovered as proteins present in tobacco plants infected with tobacco mosaic virus  <cit> .
they were defined as proteins absent in the non-infected plant and induced after pathogen infection or other adverse conditions.
pr-proteins are today classified into  <dig> families based on sequence similarity and biochemical function  <cit> .
an unusual pr family was designated pr- <dig> or intracellular pr-proteins as they are, in contrast to most pr families, expressed in the cytoplasm.
they are acidic proteins of 15– <dig> kda and were first discovered in parsley and peas in the late 1980s  <cit> .
since then, pr- <dig> family members have been found in many species of higher plants ranging from conifers to monocotyledonous and dicotyledonous angiosperms  <cit> .
in  <dig>  this protein family became a target of research in a completely different area when the major pollen allergen of white birch , bet v  <dig>  was cloned and its sequence revealed to be similar to pr- <dig> proteins   <cit> .
in subsequent years, homologous allergens from pollen of related trees such as alder and hazel as well as food allergens from fruits and vegetables such as apple and celery were identified.
immunoglobulin e cross-reactivity among these allergens is responsible for the frequent occurrence of plant food allergy among birch pollen allergic individuals, an association termed the birch-fruit syndrome  <cit> .
in  <dig>  the finding that a group of proteins abundantly expressed in the latex of opium poppy was homologous to pr- <dig> proteins despite sequence identities below 25% revealed that the pr- <dig> family was part of a much larger family of plant proteins  <cit> .
since then, several other proteins distantly related to pr- <dig> proteins were described.
-norcoclaurine synthases , enzymes involved in benzylisoquinoline alkaloid biosynthesis, from poppy  <cit>  and meadow rue   <cit>  showed sequence identities to pr- <dig> proteins between 28% and 38%  <cit> .
the sequence of a cytokinin-specific binding protein  from mung bean displayed 31% identity and 45% similarity to bet v  <dig>  <cit> .
the most striking feature of the three-dimensional structure of bet v  <dig> is the presence of a large hydrophobic cavity, which is open to the exterior and probably functions as a ligand binding site  <cit> .
the surprising similarity of the structure of the bet v 1-related major cherry allergen, pru av  <dig>  to that of the lipid binding domain of the human cholesterol-binding metastatic lymph node protein  <dig>  revealed the existence of a large superfamily of mostly lipid-binding proteins with a common fold  <cit> .
this superfamily was classified as bet v 1-like clan in the pfam protein family database  and as bet v 1-like superfamily in the structural classification of proteins  database .
in this study, we aimed, on the one hand, to classify all available sequences with significant similarity to bet v  <dig> into subfamilies and, on the other hand, to determine all structural bet v  <dig> homologues in the protein data bank  using a sensitive structural alignment tool.
these structures were classified into eleven families adding five families to the six families included in scop release  <dig> .
the ubiquitous taxonomic distribution of bet v 1-like superfamily members and their ligand binding activities suggest that the bet v  <dig> fold can be traced back to the last universal common ancestor and that the primordial bet v 1-like protein probably functioned as a lipid carrier.
RESULTS
protein families structurally related to bet v 1
in order to expand the knowledge of bet v 1-related structures beyond those already classified in scop, we performed structural alignments of representative members of all bet v 1-related families to all pdb entries.
this search yielded  <dig> structures with non-identical sequences including isoforms and engineered mutants that were classified into  <dig> families .
the structures were classified by structural similarity  and sequence similarity by performing sequence similarity searched against the uniprot and pfam databases.
in addition to the six scop families included in scop release  <dig> , five new families that contained only one or two structures were defined as members of the bet v 1-like superfamily.
anumber of structures of proteins with non-identical sequences in pdb.
broot mean square distance of backbone atoms of all aligned residues and number of aligned residues in a structural alignment with bet v  <dig> .
bet v  <dig> family
the family containing the highest number of structures was the bet v  <dig> family  with  <dig> structures with non-identical sequences.
members of this family fold into the prototypic bet v  <dig> structure with the secondary structure arrangement β-α2-β6-α with an anti-parallel β-sheet  wrapped around a long c-terminal α-helix .
subfamilies of the bet v  <dig> family  are discussed in detail below.
the pdb contained  <dig> structures of pr- <dig> subfamily members and one structure each of a major latex protein  and a csbp .
in addition, the structure of an uncharacterized protein from arabidopsis thaliana  with distant sequence similarity to mlps  can be classified as bet v 1-related, although it has a large sequence deletion resulting in the lack of two β-strands.
start family
three structures were classified into the start family , named after the steroidogenic acute regulatory  protein-related lipid transfer  domain: the start domain of the human star-related lipid transfer protein  <dig>  also termed mln <dig>  mouse star-related lipid transfer protein  <dig>  and human phosphatidylcholine transfer protein .
the structure of the start domain differs from the bet v  <dig> structure by an n-terminal extension consisting of an α-helix on the outer face of the β-sheet and two additional β-strands adding to the β-sheet on the side of the n-terminal strand .
start domains occur in a wide range of diverse, lipid-binding proteins.
pfam contained  <dig> sequences from bacteria and eukaryotes matching the start family signature .
the prototypic member of this family, star, stimulates the transport of cholesterol from the outer to the inner mitochondrial membrane, where it is required as a substrate for steroidogenic enzymes  <cit> .
other family members include ceramide transfer proteins  <cit>  and phosphatidylcholine transfer proteins  <cit> .
multi-domain proteins that contain a start domain are mammalian cytosolic acetyl-coa hydrolases  <cit>  and some members of the hd-zip family of homeodomain transcription factors from plants  <cit> .
ring hydroxylases α-chain
the pdb contained structures of  <dig> different wild-type or mutant enzymes from the aromatic ring hydroxylase family .
this family of bacterial enzymes catalyses the first step in the degradation of polycyclic aromatic compounds by converting their substrates into non-aromatic cis-dioles  <cit> .
these multi-component protein complexes consist of a ferredoxin reductase, a ferredoxin, and a hexameric hydroxylase with the subunit composition α3β <dig>  the catalytic α-subunit contains an n-terminal rieske domain with a 2fe-2s-cluster and a c-terminal bet v 1-like domain that binds a ferrous ion.
their bet v 1-like domains contain a β-sheet extended to  <dig> strands and several additional helices inserted at the n and c-termini and at an internal site .
the hydroxylases are the largest family within the bet v 1-like superfamily with  <dig> sequences recorded in pfam .
phosphatidylinositol transfer proteins
four structures were classified into the phosphatidylinositol transfer protein  family .
these eukaryotic proteins transfer phosphatidylinositol and phosphatidylcholine between membranes.
their structures differ from that of bet v  <dig> by an internal extension containing several short helices and a β-strand parallel to the first strand of the bet v 1-like sheet and a c-terminal extension that comprises a second long helix and a short stretch that closes the ligand-binding cavity in the ligand-bound conformation .
the hydrophobic ligand-binding pocket is formed by the bet v 1-like core.
the α-helical region, known as the regulatory loop, contains a protein kinase c phosphorylation site.
the c-terminus can undergo large conformational changes between a closed, ligand-bound structure, and an open, membrane bound structure that enables phospholipid binding and release  <cit> .
pfam contained  <dig> pitp sequences , most of them from animals.
mammalian pitps were classified by sequence similarity into five subfamilies, three  containing soluble single-domain proteins and two  containing large multi-domain proteins with a pitp domain at the n-terminus  <cit> .
aha <dig> family
the activator of hsp <dig> atpase homolog  <dig>  interacts with the middle domain of heat shock protein  <dig> and stimulates its atpase activity  <cit> .
its c-terminal domain is structurally related to bet v  <dig>  the pdb contained structures of eight members of the aha <dig> family , the c-terminal domain of human aha <dig> as well as one archaeal and six bacterial proteins of unknown function .
the fold of the aha1-like domain is identical to the bet v  <dig> fold except an additional n-terminal strand present in some family members, which pairs with the strand homologous to strand  <dig> in bet v  <dig> .
proteins from the aha <dig> pfam family  were found in eukaryotes, bacteria, and archaea with  <dig> sequences recorded in pfam.
while the domain organisation of most eukaryotic homologues matches that of aha <dig> suggesting a similar function, a wide range of diverse prokaryotic proteins with aha1-related domains were found in pfam.
among those were members of the arsr family, a group of metallosensitive transcription repressors  <cit> .
polyketide cyclases
the polyketide cyclase family  was named after family members with polyketide cyclase and dehydrase activity found in streptomyces species  <cit> .
the pdb contained two structures of bacterial polyketide cyclase-like predicted proteins from caulobacter crescentus and thermus thermophilus .
their folds are identical to the bet v  <dig> fold  despite low sequence identities to bet v  <dig> in a global alignment .
polyketide cyclases catalyse the cyclisation of polyketides  in the biosynthesis of a diverse group of compounds including pigments, antibiotics and anti-tumour drugs  <cit> .
there are several unrelated families of polyketide cyclases of which one is a member of the bet v 1-like superfamily .
proteins from this family are ubiquitously found in bacteria, archaea and eukaryotes forming the second largest family within the bet v 1-like superfamily with  <dig> sequences from  <dig> species, most of them proteins of unknown function, annotated in the pfam database.
the majority of the members of this family most likely possess functions other than polyketide synthesis as exemplified by the coq <dig> family of coenzyme q binding proteins from yeast and mammals .
smu440-related proteins
the structure of the predicted protein of unknown function smu <dig> from streptococcus mutans  closely matches that of bet v  <dig> , but shows only low sequence similarity .
it shares 23–46% sequence identity  with a small family of uncharacterized bacterial proteins and is classified in pfam into a non-annotated pfamb family .
pa1206-related proteins
another uncharacterized bacterial protein with a fold identical to bet v  <dig> is pa <dig> from pseudomonas aeruginosa .
it is a member of a pfam family  of proteins with unknown functions that contained  <dig> sequences from bacteria and fungi.
homotrimeric ring hydroxylases
a group of aromatic hydrocarbon hydroxylating enzymes that includes 2-oxo- <dig> -dihydroquinoline 8-monooxygenase from pseudomonas putida and carbazole  <dig> a-dioxygenase from janthinobacterium was classified into its own family .
these enzymes are homotrimers with subunits similar to the α-subunits of typical ring hydroxylating oxygenases composed of an n-terminal rieske domain that binds an iron-sulphur cluster, and a c-terminal bet v 1-like domain  <cit> .
in contrast to the typical oxygenases, they lack a β-subunit.
sequences of their c-terminal domains are only distantly related to typical oxygenases .
they do not match the ring_hydroxyl_a family signature , but a non-annotated pfamb domain .
despite similar enzymatic functions, structures of members of these families show considerable differences .
while the position of insertions to the bet v 1-like core is identical for both families , the lengths and secondary structure contents of the insertions differ.
the c-terminal extension of regular hydroxylases contains two β-strands that extend the sheet to a nine-stranded one.
in contrast, homotrimeric hydroxylases contain two additional two-stranded sheets formed by the first and second insertion.
calc-related proteins
the protein calc from micromonospora echinospora confers resistance to the enediyne antibiotic calicheamicin  <cit> .
enediynes are cytotoxic compounds whose binding to dna results in activation and oxidative cleavage of the dna strands.
calc was shown to bind calicheamicin with high affinity thereby being cleaved at a specific glycine residue, a mechanism termed self-sacrifice resistance  <cit> .
a blast search yielded 86% identity  to a predicted protein from the closely-related actinomycete salinispora arenicola and 25–36% identity  to uncharacterised bacterial members of the aha <dig> family.
the fold of calc differs from the bet v  <dig> fold by an unstructured n-terminus of  <dig> residues and the disruption of the c-terminal helix by a non-helical region of five residues in length .
coxg family
the genomes of aerobic autotrophic bacteria that utilise carbon monoxide as a carbon source contain the cox gene cluster that contains the genes encoding the subunits of the carbon monoxide dehydrogenase and several accessory genes.
one of these, coxg, is a member of the bet v 1-like superfamily.
the pdb contained two structures classified into the coxg family: predicted proteins from the bacterium geobacillus kaustophilus and the archaeon aeropyrum pernix .
their folds are identical to the bet v  <dig> fold despite the lack of significant sequence similarity .
the function of the coxg gene product was determined by gene disruption in oligotropha carboxidovorans as anchoring the co dehydrogenase to the cytoplasmic membrane  <cit> .
the pfam database  contained  <dig> sequences from bacteria and archaea, most of them predicted proteins of unknown function.
sequence similarities between members of different bet v 1-related families
members of the bet v 1-like superfamily from families other than the bet v 1-family showed sequence identities to bet v  <dig> below 25% and similarities below 40% in a global alignment .
an all-against-all comparison of representative sequences from all bet v 1-related families yielded median sequence identities below 15% and median similarities below 20% for most pairs of families .
the families whose members showed the highest degree of sequence similarity were the aha <dig> and calc families  and the polyketide cylcase and coxg families .
oxysterol binding proteins – a case of convergent evolution
the structural similarity search among all pdb entries yielded three structures of an oxysterol-binding protein from yeast .
similarly to bet v 1-like superfamily members, this protein contains a binding pocket for steroid ligands formed by a long α-helix and an anti-parallel β-sheet.
however, the topology of this structure is different with its α-helix at the n-terminus and the β-strands connected in sequential order.
the existence of this fold suggests that the structural motif of an anti-parallel β-sheet wrapped around a long α-helix is a structurally stable solution of the evolutionary challenge of developing a large, hydrophobic pocket for lipid binding.
taxonomic distribution of bet v 1-related families
ligand binding activities
the most distinctive feature of the bet v  <dig> fold is a large solvent accessible hydrophobic cavity, which may function as a ligand binding site.
in table  <dig>  experimentally determined ligands of bet v 1-like superfamily members are shown.
most ligands can be classified as bulky and hydrophobic.
the highest diversity of ligand binding activities was shown for members of the bet v  <dig> family.
the first biochemical activity proposed for pr- <dig> proteins was as ribonuclease.
rnase activity was first shown for pr- <dig> proteins from ginseng  <cit>  and later demonstrated for a number of pr- <dig> family members including bet v  <dig>  <cit> .
a possible nucleic acid binding function was supported by the fact that the most conserved part in the sequences of pr- <dig> proteins is a glycine-rich loop between strands  <dig> and  <dig>  <cit> , a so-called p-loop that is frequently found in nucleotide-binding proteins  <cit> .
however, the conformation of the glycine-rich loop differs from the canonical p-loop structure and the biological significance of the in vitro rnase activity remains controversial  <cit> .
the determination of the crystal structures of pr- <dig> proteins and their similarities with the start domain prompted researchers to search for ligands fitting into the hydrophobic cavity.
a binding activity to the plant steroid hormones brassinosteroids was experimentally determined for bet v  <dig> and pru av  <dig>  <cit> .
a different group of plant hormones, the cytokinins, was shown to bind to bet v  <dig>  <cit>  as well as to a cytokinin-binding protein from mung bean distantly related to pr- <dig>  <cit>  and a bet v  <dig> homologue from the moss physcomitrella patens  <cit> .
ligand binding activities of bet v  <dig> were extensively examined.
in addition to brassinosteroids and cytokinins, the protein was shown to bind flavonoids and fatty acids  <cit> .
the bet v  <dig> family also contains two groups of enzymes.
hyp- <dig>  a pr- <dig> from st. john's wort , catalyses the condensation of two molecules of emodin to the bioactive naphthodianthrone hypericin  <cit> .
-norcoclaurine synthases catalyse the condensation of dopamine and 4-hydroxyphenylacetaldehyde to -norcoclaurine, the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine  <cit> .
the types of ligands most frequently determined to bind other bet v 1-like superfamily members were membrane lipids .
binding of cholesterol, phospholipids and ceramide was shown for members of the start and pitp families.
a group of uncharacterised eukaryotic proteins from the polyketide cyclase family were annotated in the uniprot database as binding to coenzyme q. a function related to membrane lipid binding is binding to membranes, which is part of the biological activity of start and pitp family members, but was also shown for bet v  <dig>  <cit> .
coxg from oligotropha carboxidovorans anchors the co dehydrogenase to the cytoplasmic membrane  <cit> .
many bacterial bet v 1-related proteins exert highly specialised functions such as biosynthesis of secondary metabolites, degradation of aromatic compounds, and antibiotic resistance .
the structures of several bet v 1-related prokaryotic proteins were determined in the course of structural genomics project.
hence the biologic function of these proteins remains to be elucidated.
collection of a set of non-redundant bet v 1-related sequences
in order to gain a deeper insight into evolutionary relationships within the bet v  <dig> family, we performed a phylogenetic analysis of protein sequences with significant similarity to bet v  <dig>  the pfam database  listed  <dig> non-fragment sequences as members of the pathogenesis-related protein bet v i family .
blast database searches with representative members of already known subfamilies yielded  <dig> additional entries not listed in pfam adding up to  <dig> different bet v 1-related sequences.
this data set contained a large number of groups of highly similar isoforms and variants from the same species.
thus, the redundancy of the data set was reduced by deleting all sequences with more than 90% identity to any other sequence leaving  <dig> entries.
among those, a pr-10-like protein from the moss physcomitrella patens  and two genomic sequences from arabidopsis thaliana ,  contained two and one arabidopsis sequence  contained four bet v 1-related domains.
these proteins were split into their constituting domains.
seventeen sequences that contained large deletions were removed from the data set.
the final dataset contained  <dig> sequences or domains.
the data set contained a single sequence from outside the plant kingdom, the genomic sequence mlr <dig> from the bacterium rhizobium loti .
in order to identify further bacterial bet v  <dig> homologues, a blast search with this sequence was performed, which yielded  <dig> hits before and  <dig> sequences after reduction of redundancy using a sequence identity threshold of 90%.
this data set included three plant sequences.
due to the low sequence identity between bacterial and plant sequences , both the plant and the bacterial data set were aligned separately.
aabbreviation: csbp: cytokinin-specific binding proteins; ncs: -norcoclaurine synthases; mlp: major latex proteins; pr-10: pathogenesis-related proteins family 10; rrp: ripening-related proteins.
identification of subfamilies in the bet v  <dig> family
two data sets containing  <dig> and  <dig> sequences or domains were aligned and distance-based phylogenetic trees were constructed .
no bootstrap analysis could be performed due to the fact that about one third of the bootstrapped data sets resulted in infinite distances for some sequence pairs while the original alignment did not.
however, the branches that connected different subfamilies in the tree were generally much longer than the branches within each subfamily, thus rendering the identification of subfamilies straightforward .
eleven subfamilies of the bet v  <dig> family were identified .
plant sequences were classified into nine, bacterial sequences into two subfamilies.
the only sequence outside these taxa was the genomic sequence ddbdraft_ <dig> from the slime mould dictyostelium discoideum , which clustered with bacterial sequences .
members of all plant subfamilies but the polyketide cyclase-like subfamily matched the bet v  <dig> family signature .
the bacterial polyketide cyclase/bet v  <dig> subfamily showed no clear match with any pfam family: some members were related to the bet v  <dig> family, others could be aligned with the polyketide cyclase family signature  or the signature of a domain of unknown function .
however, the alignment scores were in most cases below the pfam gathering thresholds.
dicot pr- <dig> subfamily
the largest subfamily was the dicot pr- <dig> subfamily with  <dig> members in the non-redundant data set .
this group contained two types of proteins: genuine pr-proteins whose expression is upregulated upon pathogen infection, wounding or by abiotic stress, and pr-like proteins whose expression is developmentally regulated .
many proteins from the latter group are expressed in high amounts in pollen, seeds, fruits, or vegetative storage organs.
a striking example for this type of pr-like proteins is ocatin , the major storage protein of the tubers of the south american crop oca , which accounts for 40–60% of the total soluble protein content of the tubers  <cit> .
pr- <dig> proteins are members of small multigene families.
however, most pr- <dig> isoforms cluster exclusively with homologues from the same plant family , which can be explained either by proposing multiple independent duplication events in the common ancestors of these plant families or, more probably, by the assumption of a strong concerted evolution of pr- <dig> loci within each species.
a similar conclusion was drawn in a previous publication based on phylogenetic analysis of the much smaller number of pr- <dig> sequences known at that time  <cit> .
pr- <dig> from monocots and conifers
pr- <dig> proteins from monocots and conifers are closely related to dicot pr- <dig> proteins, both with respect to sequence similarity and function.
sequences from monocots were divided into two protein subfamilies, one restricted to cereals and one that contained members from poaceae and from other plant families such as asparagaceae and liliaceae .
subfamilies related to pr-10
two small subfamilies were more distantly related to pr- <dig> : the cytokinin-specific binding proteins  and the -norcoclaurine synthases .
members of the csbp subfamily, which is restricted to legumes, bind the plant hormone cytokinin  <cit> , an activity that has also been shown for some members of the pr- <dig> subfamily  <cit> .
proteins with ncs activity were found in thalictrum flavum  <cit>  and in opium poppy   <cit> .
two additional poppy proteins ,  as well as a protein from coptis japonica , a plant closely related to t. flavum, have been designated pr- <dig> but showed close similarities with ncs.
mlp/rrp subfamily
the second largest subfamily among plant proteins is the major latex protein/ripening-related protein  subfamily with  <dig> members,  <dig> of them from arabidopsis thaliana .
the other  <dig> sequences distribute into only  <dig> species from dicots  and monocots .
members of this subfamily were first described as proteins abundantly expressed in the latex of opium poppy   <cit> .
most members of the mlp/rrp subfamily are expressed in fruits with their expression upregulated during ripening.
examples are a ripening-induced protein from wild strawberry  <cit> , csf- <dig> from cucumber  <cit>  and sn- <dig> from bell pepper, whose expression can also be induced by wounding in unripe fruits  <cit> .
a wound-induced transcript from mesembryanthemum crystallinum  also belongs to this subfamily.
the expression of the tobacco cdna fb7- <dig> is induced during flower formation  <cit> .
the biological function of the mlp/rrp proteins is still unknown, but the expression patterns suggest a role in defence or stress response.
moss pr-10
the only bet v 1-related protein found in plants other than spermatophyta, a protein from the moss physcomitrella patents containing two bet v  <dig> domains  <cit>  shows highest similarity to the mlp/rrp subfamily .
three divergent angiosperm mlp-like sequences cluster with the p. patens domains, but contain only a single domain.
polyketide cyclase-like subfamily
a group of plant proteins distantly related to pr- <dig> as well as mlps  matched the pfam polyketide cyclase family signature .
this subfamily contains uncharacterized genomic sequences from tobacco, arabidopsis thaliana and medicago trunculata .
bacterial bet v 1-like subfamilies
bacterial bet v 1-related sequences were grouped into two subfamilies , the major one distantly related to the pfam bet v  <dig> as well as polyketide cyclase families, the minor group, which comprised only two sequences, not matching any pfam family.
most of the bacterial sequences were derived from uncharacterized genomic clones.
one exception is the periplasmic  <dig> kda protein mxad from the methylotrophic bacterium methylobacterium extorquens.
this protein enhances the rate of methanol oxidation by stimulating the interaction between methanol oxidase and its electron acceptor, cytochrome cl  <cit> .
the distant sequence similarity between plant and bacterial members of the ubiquitous polyketide cyclase family and bet v  <dig> family members suggests that the bet v  <dig> family evolved from the polyketide cyclases in plants as a response to specialized needs of higher plants with respect to development, stress response, defence, and secondary metabolism.
no clear connection between sequence and function was detected with the exception of the ncs subfamily whose members show a common function.
in contrast, hormone binding activity was found for members of the pr- <dig> and csbp families as well as for a mlp-like moss protein.
similarly, developmentally regulated, pathogen-induced and tissue-specific constitutive expression patterns are equally distributed among members of the pr- <dig> and mpl/rrp subfamilies with no correlation between phylogenetic relationship and function.
thus, the elucidation of the evolution of the bet v  <dig> family will require the identification and functional characterisation of additional bet v 1-related proteins from plants outside the spermatophyta.
CONCLUSIONS
a comparison of the structures, functions and taxonomic distributions of members of the bet v 1-like superfamily leads to the suggestion of the following evolutionary scenario .
a protein possessing the bet v  <dig> fold most likely already existed in the last universal common ancestor.
the biological function of this protein was probably related to lipid binding, such as trafficking of membrane components.
this primordial gene subsequently diverged into the multitude of bet v 1-related protein families present today, some of which retained the original fold, while others gained novel function by insertion of additional structural elements .
the ubiquitous distribution of the polyketide cyclase family and the fact that the structures of its members comprise the minimal bet v  <dig> fold without insertion of additional structural elements  suggest that these proteins are most closely related to the primordial bet v 1-like protein.
functional diversity within the bet v 1-like superfamily was also accomplished by fusion to other domains such as dna binding modules of transcription regulators found as members of several bet v 1-related families.
during evolution, sequence similarity between members of different families decreased to values that make the prediction of homology unreliable.
however, it is unlikely that this fold with the distinctive topology of the β-sheet has evolved more than once.
nevertheless, an architecture characterised by an anti-parallel β-sheet wrapped around a long α-helix forming a large cavity was invented at least twice during evolution of lipid binding proteins.
our structural similarity search showed that a family of oxysterol binding proteins adopts a fold similar to bet v  <dig> at first sight, but with a different topology.
an intriguing aspect of the evolution of the bet v 1-like superfamily is the evolution of allergenicity.
bet v  <dig> from birch pollen and possibly its close homologues from other fagales tree pollen are the only proteins within this ubiquitously distributed superfamily known to be capable of initiating an allergic immune response in humans  <cit> .
this rare occurrence of allergenicity is in sharp contrast to the distribution of allergens within the most important superfamily of allergens, the prolamins, in which numerous families of allergens were identified, such as the 2s albumins, prolamin storage proteins, non-specific lipid transfer proteins, bifunctional inhibitors, indolines, and α-globulins  <cit> .
among the still unknown factors that render a protein allergenic, structural features are frequently discussed.
hence, the comparison of bet v  <dig> with its non-allergenic structural homologues offers the possibility to shed light on such features, thus paving the way for a deeper understanding of the allergic sensitisation process and the development of novel concepts for prevention and treatment of allergy.

 plants are continuously challenged by pathogens, herbivores and adverse environmental conditions. although they lack an adaptive immune system, they have evolved numerous other mechanisms of stress response and defence. these include developmental plasticity, mechanical barriers, low molecular weight anti-microbial compounds named phytoalexins, pathogen-specific resistance genes and inducible pathogenesis-related proteins. pr-proteins were first discovered as proteins present in tobacco plants infected with tobacco mosaic virus. they were defined as proteins absent in the non-infected plant and induced after pathogen infection or other adverse conditions. pr-proteins are today classified into families based on sequence similarity and biochemical function. an unusual pr family was designated pr- or intracellular pr-proteins as they are, in contrast to most pr families, expressed in the cytoplasm. they are acidic proteins of 15– kda and were first discovered in parsley and peas in the late 1980s. since then, pr- family members have been found in many species of higher plants ranging from conifers to monocotyledonous and dicotyledonous angiosperms. in this protein family became a target of research in a completely different area when the major pollen allergen of white birch, bet v was cloned and its sequence revealed to be similar to pr- proteins. in subsequent years, homologous allergens from pollen of related trees such as alder and hazel as well as food allergens from fruits and vegetables such as apple and celery were identified. immunoglobulin e cross-reactivity among these allergens is responsible for the frequent occurrence of plant food allergy among birch pollen allergic individuals, an association termed the birch-fruit syndrome. in the finding that a group of proteins abundantly expressed in the latex of opium poppy was homologous to pr- proteins despite sequence identities below 25% revealed that the pr- family was part of a much larger family of plant proteins. since then, several other proteins distantly related to pr- proteins were described. -norcoclaurine synthases, enzymes involved in benzylisoquinoline alkaloid biosynthesis, from poppy and meadow rue showed sequence identities to pr- proteins between 28% and 38%. the sequence of a cytokinin-specific binding protein from mung bean displayed 31% identity and 45% similarity to bet v. the most striking feature of the three-dimensional structure of bet v is the presence of a large hydrophobic cavity, which is open to the exterior and probably functions as a ligand binding site. the surprising similarity of the structure of the bet v 1-related major cherry allergen, pru av to that of the lipid binding domain of the human cholesterol-binding metastatic lymph node protein revealed the existence of a large superfamily of mostly lipid-binding proteins with a common fold. this superfamily was classified as bet v 1-like clan in the pfam protein family database and as bet v 1-like superfamily in the structural classification of proteins database. in this study, we aimed, on the one hand, to classify all available sequences with significant similarity to bet v into subfamilies and, on the other hand, to determine all structural bet v homologues in the protein data bank using a sensitive structural alignment tool. these structures were classified into eleven families adding five families to the six families included in scop release. the ubiquitous taxonomic distribution of bet v 1-like superfamily members and their ligand binding activities suggest that the bet v fold can be traced back to the last universal common ancestor and that the primordial bet v 1-like protein probably functioned as a lipid carrier.  protein families structurally related to bet v 1 in order to expand the knowledge of bet v 1-related structures beyond those already classified in scop, we performed structural alignments of representative members of all bet v 1-related families to all pdb entries. this search yielded structures with non-identical sequences including isoforms and engineered mutants that were classified into families. the structures were classified by structural similarity and sequence similarity by performing sequence similarity searched against the uniprot and pfam databases. in addition to the six scop families included in scop release, five new families that contained only one or two structures were defined as members of the bet v 1-like superfamily. anumber of structures of proteins with non-identical sequences in pdb. broot mean square distance of backbone atoms of all aligned residues and number of aligned residues in a structural alignment with bet v. bet v family the family containing the highest number of structures was the bet v family with structures with non-identical sequences. members of this family fold into the prototypic bet v structure with the secondary structure arrangement β-α2-β6-α with an anti-parallel β-sheet wrapped around a long c-terminal α-helix. subfamilies of the bet v family are discussed in detail below. the pdb contained structures of pr- subfamily members and one structure each of a major latex protein and a csbp. in addition, the structure of an uncharacterized protein from arabidopsis thaliana with distant sequence similarity to mlps can be classified as bet v 1-related, although it has a large sequence deletion resulting in the lack of two β-strands. start family three structures were classified into the start family, named after the steroidogenic acute regulatory protein-related lipid transfer domain: the start domain of the human star-related lipid transfer protein also termed mln mouse star-related lipid transfer protein and human phosphatidylcholine transfer protein. the structure of the start domain differs from the bet v structure by an n-terminal extension consisting of an α-helix on the outer face of the β-sheet and two additional β-strands adding to the β-sheet on the side of the n-terminal strand. start domains occur in a wide range of diverse, lipid-binding proteins. pfam contained sequences from bacteria and eukaryotes matching the start family signature. the prototypic member of this family, star, stimulates the transport of cholesterol from the outer to the inner mitochondrial membrane, where it is required as a substrate for steroidogenic enzymes. other family members include ceramide transfer proteins and phosphatidylcholine transfer proteins. multi-domain proteins that contain a start domain are mammalian cytosolic acetyl-coa hydrolases and some members of the hd-zip family of homeodomain transcription factors from plants. ring hydroxylases α-chain the pdb contained structures of different wild-type or mutant enzymes from the aromatic ring hydroxylase family. this family of bacterial enzymes catalyses the first step in the degradation of polycyclic aromatic compounds by converting their substrates into non-aromatic cis-dioles. these multi-component protein complexes consist of a ferredoxin reductase, a ferredoxin, and a hexameric hydroxylase with the subunit composition α3β the catalytic α-subunit contains an n-terminal rieske domain with a 2fe-2s-cluster and a c-terminal bet v 1-like domain that binds a ferrous ion. their bet v 1-like domains contain a β-sheet extended to strands and several additional helices inserted at the n and c-termini and at an internal site. the hydroxylases are the largest family within the bet v 1-like superfamily with sequences recorded in pfam. phosphatidylinositol transfer proteins four structures were classified into the phosphatidylinositol transfer protein family. these eukaryotic proteins transfer phosphatidylinositol and phosphatidylcholine between membranes. their structures differ from that of bet v by an internal extension containing several short helices and a β-strand parallel to the first strand of the bet v 1-like sheet and a c-terminal extension that comprises a second long helix and a short stretch that closes the ligand-binding cavity in the ligand-bound conformation. the hydrophobic ligand-binding pocket is formed by the bet v 1-like core. the α-helical region, known as the regulatory loop, contains a protein kinase c phosphorylation site. the c-terminus can undergo large conformational changes between a closed, ligand-bound structure, and an open, membrane bound structure that enables phospholipid binding and release. pfam contained pitp sequences, most of them from animals. mammalian pitps were classified by sequence similarity into five subfamilies, three containing soluble single-domain proteins and two containing large multi-domain proteins with a pitp domain at the n-terminus. aha family the activator of hsp atpase homolog interacts with the middle domain of heat shock protein and stimulates its atpase activity. its c-terminal domain is structurally related to bet v the pdb contained structures of eight members of the aha family, the c-terminal domain of human aha as well as one archaeal and six bacterial proteins of unknown function. the fold of the aha1-like domain is identical to the bet v fold except an additional n-terminal strand present in some family members, which pairs with the strand homologous to strand in bet v. proteins from the aha pfam family were found in eukaryotes, bacteria, and archaea with sequences recorded in pfam. while the domain organisation of most eukaryotic homologues matches that of aha suggesting a similar function, a wide range of diverse prokaryotic proteins with aha1-related domains were found in pfam. among those were members of the arsr family, a group of metallosensitive transcription repressors. polyketide cyclases the polyketide cyclase family was named after family members with polyketide cyclase and dehydrase activity found in streptomyces species. the pdb contained two structures of bacterial polyketide cyclase-like predicted proteins from caulobacter crescentus and thermus thermophilus. their folds are identical to the bet v fold despite low sequence identities to bet v in a global alignment. polyketide cyclases catalyse the cyclisation of polyketides in the biosynthesis of a diverse group of compounds including pigments, antibiotics and anti-tumour drugs. there are several unrelated families of polyketide cyclases of which one is a member of the bet v 1-like superfamily. proteins from this family are ubiquitously found in bacteria, archaea and eukaryotes forming the second largest family within the bet v 1-like superfamily with sequences from species, most of them proteins of unknown function, annotated in the pfam database. the majority of the members of this family most likely possess functions other than polyketide synthesis as exemplified by the coq family of coenzyme q binding proteins from yeast and mammals. smu440-related proteins the structure of the predicted protein of unknown function smu from streptococcus mutans closely matches that of bet v, but shows only low sequence similarity. it shares 23–46% sequence identity with a small family of uncharacterized bacterial proteins and is classified in pfam into a non-annotated pfamb family. pa1206-related proteins another uncharacterized bacterial protein with a fold identical to bet v is pa from pseudomonas aeruginosa. it is a member of a pfam family of proteins with unknown functions that contained sequences from bacteria and fungi. homotrimeric ring hydroxylases a group of aromatic hydrocarbon hydroxylating enzymes that includes 2-oxo- -dihydroquinoline 8-monooxygenase from pseudomonas putida and carbazole a-dioxygenase from janthinobacterium was classified into its own family. these enzymes are homotrimers with subunits similar to the α-subunits of typical ring hydroxylating oxygenases composed of an n-terminal rieske domain that binds an iron-sulphur cluster, and a c-terminal bet v 1-like domain. in contrast to the typical oxygenases, they lack a β-subunit. sequences of their c-terminal domains are only distantly related to typical oxygenases. they do not match the ring_hydroxyl_a family signature, but a non-annotated pfamb domain. despite similar enzymatic functions, structures of members of these families show considerable differences. while the position of insertions to the bet v 1-like core is identical for both families, the lengths and secondary structure contents of the insertions differ. the c-terminal extension of regular hydroxylases contains two β-strands that extend the sheet to a nine-stranded one. in contrast, homotrimeric hydroxylases contain two additional two-stranded sheets formed by the first and second insertion. calc-related proteins the protein calc from micromonospora echinospora confers resistance to the enediyne antibiotic calicheamicin. enediynes are cytotoxic compounds whose binding to dna results in activation and oxidative cleavage of the dna strands. calc was shown to bind calicheamicin with high affinity thereby being cleaved at a specific glycine residue, a mechanism termed self-sacrifice resistance. a blast search yielded 86% identity to a predicted protein from the closely-related actinomycete salinispora arenicola and 25–36% identity to uncharacterised bacterial members of the aha family. the fold of calc differs from the bet v fold by an unstructured n-terminus of residues and the disruption of the c-terminal helix by a non-helical region of five residues in length. coxg family the genomes of aerobic autotrophic bacteria that utilise carbon monoxide as a carbon source contain the cox gene cluster that contains the genes encoding the subunits of the carbon monoxide dehydrogenase and several accessory genes. one of these, coxg, is a member of the bet v 1-like superfamily. the pdb contained two structures classified into the coxg family: predicted proteins from the bacterium geobacillus kaustophilus and the archaeon aeropyrum pernix. their folds are identical to the bet v fold despite the lack of significant sequence similarity. the function of the coxg gene product was determined by gene disruption in oligotropha carboxidovorans as anchoring the co dehydrogenase to the cytoplasmic membrane. the pfam database contained sequences from bacteria and archaea, most of them predicted proteins of unknown function. sequence similarities between members of different bet v 1-related families members of the bet v 1-like superfamily from families other than the bet v 1-family showed sequence identities to bet v below 25% and similarities below 40% in a global alignment. an all-against-all comparison of representative sequences from all bet v 1-related families yielded median sequence identities below 15% and median similarities below 20% for most pairs of families. the families whose members showed the highest degree of sequence similarity were the aha and calc families and the polyketide cylcase and coxg families. oxysterol binding proteins – a case of convergent evolution the structural similarity search among all pdb entries yielded three structures of an oxysterol-binding protein from yeast. similarly to bet v 1-like superfamily members, this protein contains a binding pocket for steroid ligands formed by a long α-helix and an anti-parallel β-sheet. however, the topology of this structure is different with its α-helix at the n-terminus and the β-strands connected in sequential order. the existence of this fold suggests that the structural motif of an anti-parallel β-sheet wrapped around a long α-helix is a structurally stable solution of the evolutionary challenge of developing a large, hydrophobic pocket for lipid binding. taxonomic distribution of bet v 1-related families ligand binding activities the most distinctive feature of the bet v fold is a large solvent accessible hydrophobic cavity, which may function as a ligand binding site. in table experimentally determined ligands of bet v 1-like superfamily members are shown. most ligands can be classified as bulky and hydrophobic. the highest diversity of ligand binding activities was shown for members of the bet v family. the first biochemical activity proposed for pr- proteins was as ribonuclease. rnase activity was first shown for pr- proteins from ginseng and later demonstrated for a number of pr- family members including bet v. a possible nucleic acid binding function was supported by the fact that the most conserved part in the sequences of pr- proteins is a glycine-rich loop between strands and, a so-called p-loop that is frequently found in nucleotide-binding proteins. however, the conformation of the glycine-rich loop differs from the canonical p-loop structure and the biological significance of the in vitro rnase activity remains controversial. the determination of the crystal structures of pr- proteins and their similarities with the start domain prompted researchers to search for ligands fitting into the hydrophobic cavity. a binding activity to the plant steroid hormones brassinosteroids was experimentally determined for bet v and pru av. a different group of plant hormones, the cytokinins, was shown to bind to bet v as well as to a cytokinin-binding protein from mung bean distantly related to pr- and a bet v homologue from the moss physcomitrella patens. ligand binding activities of bet v were extensively examined. in addition to brassinosteroids and cytokinins, the protein was shown to bind flavonoids and fatty acids. the bet v family also contains two groups of enzymes. hyp- a pr- from st. john's wort, catalyses the condensation of two molecules of emodin to the bioactive naphthodianthrone hypericin. -norcoclaurine synthases catalyse the condensation of dopamine and 4-hydroxyphenylacetaldehyde to -norcoclaurine, the first committed step in the biosynthesis of benzylisoquinoline alkaloids such as morphine. the types of ligands most frequently determined to bind other bet v 1-like superfamily members were membrane lipids. binding of cholesterol, phospholipids and ceramide was shown for members of the start and pitp families. a group of uncharacterised eukaryotic proteins from the polyketide cyclase family were annotated in the uniprot database as binding to coenzyme q. a function related to membrane lipid binding is binding to membranes, which is part of the biological activity of start and pitp family members, but was also shown for bet v. coxg from oligotropha carboxidovorans anchors the co dehydrogenase to the cytoplasmic membrane. many bacterial bet v 1-related proteins exert highly specialised functions such as biosynthesis of secondary metabolites, degradation of aromatic compounds, and antibiotic resistance. the structures of several bet v 1-related prokaryotic proteins were determined in the course of structural genomics project. hence the biologic function of these proteins remains to be elucidated. collection of a set of non-redundant bet v 1-related sequences in order to gain a deeper insight into evolutionary relationships within the bet v family, we performed a phylogenetic analysis of protein sequences with significant similarity to bet v the pfam database listed non-fragment sequences as members of the pathogenesis-related protein bet v i family. blast database searches with representative members of already known subfamilies yielded additional entries not listed in pfam adding up to different bet v 1-related sequences. this data set contained a large number of groups of highly similar isoforms and variants from the same species. thus, the redundancy of the data set was reduced by deleting all sequences with more than 90% identity to any other sequence leaving entries. among those, a pr-10-like protein from the moss physcomitrella patens and two genomic sequences from arabidopsis thaliana, contained two and one arabidopsis sequence contained four bet v 1-related domains. these proteins were split into their constituting domains. seventeen sequences that contained large deletions were removed from the data set. the final dataset contained sequences or domains. the data set contained a single sequence from outside the plant kingdom, the genomic sequence mlr from the bacterium rhizobium loti. in order to identify further bacterial bet v homologues, a blast search with this sequence was performed, which yielded hits before and sequences after reduction of redundancy using a sequence identity threshold of 90%. this data set included three plant sequences. due to the low sequence identity between bacterial and plant sequences, both the plant and the bacterial data set were aligned separately. aabbreviation: csbp: cytokinin-specific binding proteins; ncs: -norcoclaurine synthases; mlp: major latex proteins; pr-10: pathogenesis-related proteins family 10; rrp: ripening-related proteins. identification of subfamilies in the bet v family two data sets containing and sequences or domains were aligned and distance-based phylogenetic trees were constructed. no bootstrap analysis could be performed due to the fact that about one third of the bootstrapped data sets resulted in infinite distances for some sequence pairs while the original alignment did not. however, the branches that connected different subfamilies in the tree were generally much longer than the branches within each subfamily, thus rendering the identification of subfamilies straightforward. eleven subfamilies of the bet v family were identified. plant sequences were classified into nine, bacterial sequences into two subfamilies. the only sequence outside these taxa was the genomic sequence ddbdraft_ from the slime mould dictyostelium discoideum, which clustered with bacterial sequences. members of all plant subfamilies but the polyketide cyclase-like subfamily matched the bet v family signature. the bacterial polyketide cyclase/bet v subfamily showed no clear match with any pfam family: some members were related to the bet v family, others could be aligned with the polyketide cyclase family signature or the signature of a domain of unknown function. however, the alignment scores were in most cases below the pfam gathering thresholds. dicot pr- subfamily the largest subfamily was the dicot pr- subfamily with members in the non-redundant data set. this group contained two types of proteins: genuine pr-proteins whose expression is upregulated upon pathogen infection, wounding or by abiotic stress, and pr-like proteins whose expression is developmentally regulated. many proteins from the latter group are expressed in high amounts in pollen, seeds, fruits, or vegetative storage organs. a striking example for this type of pr-like proteins is ocatin, the major storage protein of the tubers of the south american crop oca, which accounts for 40–60% of the total soluble protein content of the tubers. pr- proteins are members of small multigene families. however, most pr- isoforms cluster exclusively with homologues from the same plant family, which can be explained either by proposing multiple independent duplication events in the common ancestors of these plant families or, more probably, by the assumption of a strong concerted evolution of pr- loci within each species. a similar conclusion was drawn in a previous publication based on phylogenetic analysis of the much smaller number of pr- sequences known at that time. pr- from monocots and conifers pr- proteins from monocots and conifers are closely related to dicot pr- proteins, both with respect to sequence similarity and function. sequences from monocots were divided into two protein subfamilies, one restricted to cereals and one that contained members from poaceae and from other plant families such as asparagaceae and liliaceae. subfamilies related to pr-10 two small subfamilies were more distantly related to pr-: the cytokinin-specific binding proteins and the -norcoclaurine synthases. members of the csbp subfamily, which is restricted to legumes, bind the plant hormone cytokinin, an activity that has also been shown for some members of the pr- subfamily. proteins with ncs activity were found in thalictrum flavum and in opium poppy. two additional poppy proteins, as well as a protein from coptis japonica, a plant closely related to t. flavum, have been designated pr- but showed close similarities with ncs. mlp/rrp subfamily the second largest subfamily among plant proteins is the major latex protein/ripening-related protein subfamily with members, of them from arabidopsis thaliana. the other sequences distribute into only species from dicots and monocots. members of this subfamily were first described as proteins abundantly expressed in the latex of opium poppy. most members of the mlp/rrp subfamily are expressed in fruits with their expression upregulated during ripening. examples are a ripening-induced protein from wild strawberry, csf- from cucumber and sn- from bell pepper, whose expression can also be induced by wounding in unripe fruits. a wound-induced transcript from mesembryanthemum crystallinum also belongs to this subfamily. the expression of the tobacco cdna fb7- is induced during flower formation. the biological function of the mlp/rrp proteins is still unknown, but the expression patterns suggest a role in defence or stress response. moss pr-10 the only bet v 1-related protein found in plants other than spermatophyta, a protein from the moss physcomitrella patents containing two bet v domains shows highest similarity to the mlp/rrp subfamily. three divergent angiosperm mlp-like sequences cluster with the p. patens domains, but contain only a single domain. polyketide cyclase-like subfamily a group of plant proteins distantly related to pr- as well as mlps matched the pfam polyketide cyclase family signature. this subfamily contains uncharacterized genomic sequences from tobacco, arabidopsis thaliana and medicago trunculata. bacterial bet v 1-like subfamilies bacterial bet v 1-related sequences were grouped into two subfamilies, the major one distantly related to the pfam bet v as well as polyketide cyclase families, the minor group, which comprised only two sequences, not matching any pfam family. most of the bacterial sequences were derived from uncharacterized genomic clones. one exception is the periplasmic kda protein mxad from the methylotrophic bacterium methylobacterium extorquens. this protein enhances the rate of methanol oxidation by stimulating the interaction between methanol oxidase and its electron acceptor, cytochrome cl. the distant sequence similarity between plant and bacterial members of the ubiquitous polyketide cyclase family and bet v family members suggests that the bet v family evolved from the polyketide cyclases in plants as a response to specialized needs of higher plants with respect to development, stress response, defence, and secondary metabolism. no clear connection between sequence and function was detected with the exception of the ncs subfamily whose members show a common function. in contrast, hormone binding activity was found for members of the pr- and csbp families as well as for a mlp-like moss protein. similarly, developmentally regulated, pathogen-induced and tissue-specific constitutive expression patterns are equally distributed among members of the pr- and mpl/rrp subfamilies with no correlation between phylogenetic relationship and function. thus, the elucidation of the evolution of the bet v family will require the identification and functional characterisation of additional bet v 1-related proteins from plants outside the spermatophyta.  a comparison of the structures, functions and taxonomic distributions of members of the bet v 1-like superfamily leads to the suggestion of the following evolutionary scenario. a protein possessing the bet v fold most likely already existed in the last universal common ancestor. the biological function of this protein was probably related to lipid binding, such as trafficking of membrane components. this primordial gene subsequently diverged into the multitude of bet v 1-related protein families present today, some of which retained the original fold, while others gained novel function by insertion of additional structural elements. the ubiquitous distribution of the polyketide cyclase family and the fact that the structures of its members comprise the minimal bet v fold without insertion of additional structural elements suggest that these proteins are most closely related to the primordial bet v 1-like protein. functional diversity within the bet v 1-like superfamily was also accomplished by fusion to other domains such as dna binding modules of transcription regulators found as members of several bet v 1-related families. during evolution, sequence similarity between members of different families decreased to values that make the prediction of homology unreliable. however, it is unlikely that this fold with the distinctive topology of the β-sheet has evolved more than once. nevertheless, an architecture characterised by an anti-parallel β-sheet wrapped around a long α-helix forming a large cavity was invented at least twice during evolution of lipid binding proteins. our structural similarity search showed that a family of oxysterol binding proteins adopts a fold similar to bet v at first sight, but with a different topology. an intriguing aspect of the evolution of the bet v 1-like superfamily is the evolution of allergenicity. bet v from birch pollen and possibly its close homologues from other fagales tree pollen are the only proteins within this ubiquitously distributed superfamily known to be capable of initiating an allergic immune response in humans. this rare occurrence of allergenicity is in sharp contrast to the distribution of allergens within the most important superfamily of allergens, the prolamins, in which numerous families of allergens were identified, such as the 2s albumins, prolamin storage proteins, non-specific lipid transfer proteins, bifunctional inhibitors, indolines, and α-globulins. among the still unknown factors that render a protein allergenic, structural features are frequently discussed. hence, the comparison of bet v with its non-allergenic structural homologues offers the possibility to shed light on such features, thus paving the way for a deeper understanding of the allergic sensitisation process and the development of novel concepts for prevention and treatment of allergy. 
in this study, we aimed to identify and classify all bet v 1-related structures from the protein data bank and all bet v 1-related sequences from the uniprot database. structural comparisons of representative members of already known protein families structurally related to bet v with all entries of the protein data bank yielded structures with non-identical sequences. the ubiquitous distribution of bet v 1-related proteins among all superkingdoms suggests that a bet v 1-like protein was already present in the last universal common ancestor. the taxonomic distribution of these families extracted from the pfam protein family database showed that members of the polyketide cyclase family and the activator of hsp atpase homologue family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. they were classified into eleven families, five of which were newly identified and not included in the structural classification of proteins database release. plant sequences included the pathogenesis-related proteins the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences. a distance-based phylogenetic tree yielded a classification into subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. during evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands. comparison of ligand binding activities of bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. phylogenetic relationships within the bet v family, defined as the group of proteins with significant sequence similarity to bet v were determined by aligning bet v 1-related sequences.
 the major birch pollen allergen, bet v is a member of the ubiquitous pr- family of plant pathogenesis-related proteins. in recent years, a number of diverse plant proteins with low sequence similarity to bet v was identified. in addition, determination of the bet v structure revealed the existence of a large superfamily of structurally related proteins. in this study, we aimed to identify and classify all bet v 1-related structures from the protein data bank and all bet v 1-related sequences from the uniprot database.  structural comparisons of representative members of already known protein families structurally related to bet v with all entries of the protein data bank yielded structures with non-identical sequences. they were classified into eleven families, five of which were newly identified and not included in the structural classification of proteins database release. the taxonomic distribution of these families extracted from the pfam protein family database showed that members of the polyketide cyclase family and the activator of hsp atpase homologue family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. comparison of ligand binding activities of bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. phylogenetic relationships within the bet v family, defined as the group of proteins with significant sequence similarity to bet v were determined by aligning bet v 1-related sequences. a distance-based phylogenetic tree yielded a classification into subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. plant sequences included the pathogenesis-related proteins the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.  the ubiquitous distribution of bet v 1-related proteins among all superkingdoms suggests that a bet v 1-like protein was already present in the last universal common ancestor. during evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands. 