BACKGROUND
lipases  are a versatile group of enzymes which catalyze the hydrolysis or synthesis of a broad range of water insoluble esters.
they belong to the class of α/β-hydrolases which also contains esterases, acetylcholinesterases, cutinases, carboxylesterases and epoxide hydrolases.
despite their high diversity in sequence and function, the α/β-hydrolases share a common architecture, the α/β-hydrolase fold  <cit>  and conserved active site signatures, the gxsxg and gxdxg motifs  <cit> .
two conserved features found in all α/β-hydrolases are the active site, consisting of the catalytic triad of s-d-h, and the oxyanion hole.
depending on the amino acids involved in forming the oxyanion hole, the enzymes can be classified into three classes, the gggx-, gx-, and the y-class  <cit> .
the lipase engineering database   <cit>  is a resource of fully and consistently annotated superfamilies and homologous families of α/β hydrolases including multisequence alignments of all families.
the curation and annotation process for the led is supported by dwarf  <cit> , an inhouse data warehouse system for protein families.
the led is accessible by a web interface at http://www.led.uni-stuttgart.de.
it can be browsed on the level of families, organisms, or structures, and blast searches can be performed against all sequence entries.
prominent members of the α/β hydrolases are the two lipases from candida antarctica.
lipase b is a versatile and well characterized biocatalyst in many organic syntheses and biotransformations  <cit>  and shows a low sequence similarity to other α/β hydrolases.
the second lipase from candida antarctica, lipase a , shows a number of unique biocatalytic properties among hydrolases, e.g.
high thermostability and stability at acidic ph ranges and the acceptance of tertiary and sterically hindered alcohols  <cit> .
cala also has a low sequence similarity to other members of the α/β hydrolase fold including lipase b. therefore it was not included in previous versions of the led.
only after its structure was recently determined  <cit> , a detailed analysis of its structure identified cala unambiguously as a member of the α/β hydrolase family.
however, in this structure the active site is not accessible to a substrate, therefore the molecular details of substrate binding or the existence of a possible lid are still elusive.
RESULTS
database content and layout
release  <dig>  of the lipase engineering database  contains  <dig> proteins with  <dig> sequence and  <dig> structure entries of which about  <dig> protein and  <dig> structure entries are new.
six new homologous families and one new superfamily  have been added to the led in the update process.
seed sequences for the new "candida antarctica lipase a like" superfamily  included the sequence from the resolved crystal structure  and three sequences of homologous lipases from other organisms  which showed high sequence similarity to candida antarctica lipase a   <cit> .
most of the sequences of the superfamily abh <dig> have already been assigned to a common protein family in other protein family databases.
in the pfam database  <cit>  they are included in the lip  family, in the interpro database  <cit>  in the family ipr <dig>  and in the esther  <cit>  database in the fungal-bact_lip family.
because we included only sequences of high sequence similarity to guarantee a good alignment of all sequences of individual families, especially of active site residues, our family abh <dig> contains less sequences than the respective protein families of the other databases.
the four largest superfamilies in release  <dig>  contain 50% of all proteins in the led: the "cytosolic hydrolases" superfamily  with  <dig> proteins, containing epoxide hydrolases and haloalkane dehalogenases, the "carboxylesterases" superfamily  with  <dig> proteins, containing a wide range of carboxylesterases, such as acetylcholine esterases and bile salt activated lipases, the "moraxella lipase  <dig> like" superfamily  with  <dig> proteins containing mainly lipases and carboxylesterases, and the "microsomal hydrolases" superfamily  with  <dig> proteins, containing microsomal epoxide hydrolases and peptidases.
the "cytosolic hydrolases" and "microsomal hydrolases" superfamilies  belong to the gx-class of α/β hydrolases, the "carboxylesterases" and "moraxella lipase  <dig> like" superfamilies  belong to the gggx-class of α/β hydrolases.
candida antarctica lipase a protein family
the "candida antarctica lipase a like" superfamily contains one crystal structure and  <dig> sequences, assigned to  <dig> proteins.
they were grouped into four homologous families based on sequence similarity.
homologous families were named by the organism of origin of the dominating proteins in the respective family : the "candida antarctica lipase a like" homologous family consisting of lipase a from c. antarctica, the "malassezia lipase like" homologous family consisting entirely of lipases and esterases from malassezia globosa or malassezia furfur, the "candida albicans lipase like" homologous family consisting of various isoforms of the secretory lipase from candida albicans, and the "aspergillus lipase like" homologous family consisting mainly of hypothetical or putative lipases, mostly from aspergillus.
all  <dig> proteins are from organisms belonging to the subkingdom dikarya of the kingdom fungi.
<dig> proteins are classified as either lipases or esterases in genbank  <cit>  while  <dig> proteins are classified as putative or hypothetical.
the only structure entry in this superfamily is from the recently resolved crystal structure of cala  <cit> .
based on the structure of the oxyanion hole, cala can be classified as a y-class lipase, and tyr  <dig> was identified as the oxyanion hole forming amino acid.
a structural comparison with other structures from the led identified a structure from the deacetylase superfamily  as most closely related.
a detailed structural alignment of cala  with the structure of the bacillus subtilis deacetylase  from the deacetylase superfamily showed a superimposition of the common α/β hydrolase fold including the catalytic triad , despite having a low overall sequence identity of only 15%.
structural differences between the two structures are found in the cap and the c-terminal region.
the cap region, located between β-strands  <dig> and  <dig>  often confers substrate specificity or additional functions to the enzyme  <cit> .
in the case of cala, the cap region is involved in forming the tunnel like binding site for the acyl moiety  <cit> .
for b. subtilis deacetylase, the cap region partially shields the active site from the solvent  <cit> .
the cap region of cala consists of six α-helices, while the cap region of b. subtilis deacetylase consists of only four α-helices .
three α-helices in both proteins are found at identical positions.
cala shows an insert of three additional α-helices after the first two conserved α-helices and is missing the last α-helix of the cap present in b. subtilis deacetylase .
in order to identify residues in the cap region which are conserved between the "candida antarctica lipase a like" and "deacetylases" superfamilies, a multisequence alignment of each family was performed.
the two family alignments were aligned using a structural alignment of the two protein structures .
the alignment demonstrated that despite the high structural similarity, there are no conserved residues in the cap region of both protein families.
two hidden markov models of the three inserted α-helices of cala and the α-helices shared by both proteins were created and used to search against all other protein families of the led.
no sequences with a significant similarity were found in the entire database, demonstrating that the sequence of the cap region of the "candida antarctica lipase a like" superfamily is unique.
in comparison to the b. subtilis deacetylase, cala has two additional β-strands  in the c-terminal region .
they are positioned directly above the active site and prevent a direct access of the substrate to the active site.
we assume that the β-strands  <dig> and  <dig> perform a lid like function for cala since movement of the two β-strands would allow substrate access to the active site of cala from a similar direction as for the b. subtilis deacetylases  <cit> .
a comparison of the substrate binding sites of both proteins showed that the alcohol binding site is similar in both proteins and provides ample space for alcohol moieties of substrates .
therefore, both proteins are expected to accept a variety of bulky alcohols.
the binding sites for the acyl moieties are highly different.
cala has a long, tunnel like binding site, while the b. subtilis deacetylase has a small cavity which is part of a cleft on the protein surface.
therefore, the acyl moieties of the substrates are expected to differ significantly between both enzymes.
cala is expected to accept mediumt to long chain fatty acids, while the b. subtilis deacetylase is limited to short-chain acyl moieties.
thus, despite the overall similarity between cala and the b. subtilis deacetylase, the acyl binding site is fundamentally different.
however, the binding site of cala shows surprising similarity to another lipase, candida rugosa lipase .
for crl, two different structural confirmations have been resolved, an open conformation   <cit> , and a closed conformation   <cit>  where the lid of crl is blocking the substrate access to the active site.
crl has a cap region between β-strands  <dig> and  <dig>  consisting of four α-helices .
the substrate binding site of crl consists of a long tunnel for the acyl moiety of the substrate and provides ample space for the alcohol moiety of the substrate .
despite having a lower overall structure similarity to cala than the b. subtilis deacetylase, the binding sites of cala and crl are highly similar .
both provide space for large, bulky alcohol moieties of the substrate and have a tunnel like binding site for the acyl moiety.
both proteins posses a lid which covers the active site and prevents direct access to the substrate binding site in its closed state.
the lid of crl lipase is formed by a α-helix between β-strands  <dig> and  <dig> and is located in the n-terminal region while the putative lid in cala is formed by the two c-terminal β-strands  <dig> and  <dig> .
discussion
the led contains annotated and systematically classified protein families of α/β hydrolases.
it has been shown to be a useful tool for the systematic analysis of protein families.
previous work employed the led and blast in order to identify novel enzymes belonging to the α/β hydrolase fold  <cit> .
a model for the prediction of protein solubility was developed and refined by performing a comprehensive analysis of the protein families of the led  <cit> .
a further study involved the systematic analysis of protein families of the led in regard to the distribution and conservation of functionally relevant rare codons  <cit> .
since the first release of the led  <cit> , more than  <dig> new α/β hydrolases became available and were integrated in the release  <dig> .
as a case study for the utility of the highly enriched and annotated database, the newly introduced superfamily of cala was analysed and compared to other protein structures in the led.
the goal was to characterise the sequence and structure of cala in comparison to other α/β hydrolases despite its low sequence similarity and to understand the molecular basis of substrate recognition.
while cala shows structural similarity to the deacetylase family, the substrate specificity of both enzymes differs, which is consistent with the differences observed in the substrate binding sites of both proteins.
in contrast, the lipase from c. rugosa , which shows a lower overall structural similarity to cala, is remarkably similar in regard to the substrate binding site.
the structural similarities and differences are in accordance with experimentally observed substrate specificities of the three enzymes.
all three proteins have a spacious alcohol binding site.
the b. subtilis deacetylase accepts a wide variety of bulky substrates like cephalosporin c and xylose  <cit> .
cala and crl also accept bulky substrates, ranging from primary alcohols to sterically hindered secondary alcohols and even tertiary alcohols  <cit> .
the tunnel like binding site of cala allows the enzyme to accept esters of long chain fatty acids  <cit> .
the similar tunnel like acyl binding site of crl also accepts fatty acids up to a chain length of  <dig>  <cit> .
in contrast, the small acyl binding site of the b. subtilis deacetylase is unable to accept large acyl groups and is restricted towards acetyl moieties  <cit> .
experimentally, cala and crl have been shown to display interfacial activation  <cit> .
while a lid in crl has been localized and the open and closed form of crl has been crystallized , the lid function of the β-strands 9- <dig> in cala remains to be experimentally verified.
however, the similarities to crl suggest a substrate access involving the movement of β-strands 9- <dig>
CONCLUSIONS
the analysis of the newly introduced protein family of candida antarctica lipase a demonstrates the strength of our database approach by providing a large set of protein families which share a common protein fold despite an overall low sequence similarity.
by combining both, structural and sequential information of a large number of proteins a thorough analysis and classification of proteins of interest is made possible.
the lipase engineering database  is online accessible at http://www.led.uni-stuttgart.de.
all information on families of sequence and structure data, as well as alignments, phylogenetic trees, and family-specific profiles can be accessed by manual download.

 lipases are a versatile group of enzymes which catalyze the hydrolysis or synthesis of a broad range of water insoluble esters. they belong to the class of α/β-hydrolases which also contains esterases, acetylcholinesterases, cutinases, carboxylesterases and epoxide hydrolases. despite their high diversity in sequence and function, the α/β-hydrolases share a common architecture, the α/β-hydrolase fold and conserved active site signatures, the gxsxg and gxdxg motifs. two conserved features found in all α/β-hydrolases are the active site, consisting of the catalytic triad of s-d-h, and the oxyanion hole. depending on the amino acids involved in forming the oxyanion hole, the enzymes can be classified into three classes, the gggx-, gx-, and the y-class. the lipase engineering database is a resource of fully and consistently annotated superfamilies and homologous families of α/β hydrolases including multisequence alignments of all families. the curation and annotation process for the led is supported by dwarf, an inhouse data warehouse system for protein families. the led is accessible by a web interface at http://www.led.uni-stuttgart.de. it can be browsed on the level of families, organisms, or structures, and blast searches can be performed against all sequence entries. prominent members of the α/β hydrolases are the two lipases from candida antarctica. lipase b is a versatile and well characterized biocatalyst in many organic syntheses and biotransformations and shows a low sequence similarity to other α/β hydrolases. the second lipase from candida antarctica, lipase a, shows a number of unique biocatalytic properties among hydrolases, e.g. high thermostability and stability at acidic ph ranges and the acceptance of tertiary and sterically hindered alcohols. cala also has a low sequence similarity to other members of the α/β hydrolase fold including lipase b. therefore it was not included in previous versions of the led. only after its structure was recently determined, a detailed analysis of its structure identified cala unambiguously as a member of the α/β hydrolase family. however, in this structure the active site is not accessible to a substrate, therefore the molecular details of substrate binding or the existence of a possible lid are still elusive.  database content and layout release of the lipase engineering database contains proteins with sequence and structure entries of which about protein and structure entries are new. six new homologous families and one new superfamily have been added to the led in the update process. seed sequences for the new "candida antarctica lipase a like" superfamily included the sequence from the resolved crystal structure and three sequences of homologous lipases from other organisms which showed high sequence similarity to candida antarctica lipase a. most of the sequences of the superfamily abh have already been assigned to a common protein family in other protein family databases. in the pfam database they are included in the lip family, in the interpro database in the family ipr and in the esther database in the fungal-bact_lip family. because we included only sequences of high sequence similarity to guarantee a good alignment of all sequences of individual families, especially of active site residues, our family abh contains less sequences than the respective protein families of the other databases. the four largest superfamilies in release contain 50% of all proteins in the led: the "cytosolic hydrolases" superfamily with proteins, containing epoxide hydrolases and haloalkane dehalogenases, the "carboxylesterases" superfamily with proteins, containing a wide range of carboxylesterases, such as acetylcholine esterases and bile salt activated lipases, the "moraxella lipase like" superfamily with proteins containing mainly lipases and carboxylesterases, and the "microsomal hydrolases" superfamily with proteins, containing microsomal epoxide hydrolases and peptidases. the "cytosolic hydrolases" and "microsomal hydrolases" superfamilies belong to the gx-class of α/β hydrolases, the "carboxylesterases" and "moraxella lipase like" superfamilies belong to the gggx-class of α/β hydrolases. candida antarctica lipase a protein family the "candida antarctica lipase a like" superfamily contains one crystal structure and sequences, assigned to proteins. they were grouped into four homologous families based on sequence similarity. homologous families were named by the organism of origin of the dominating proteins in the respective family: the "candida antarctica lipase a like" homologous family consisting of lipase a from c. antarctica, the "malassezia lipase like" homologous family consisting entirely of lipases and esterases from malassezia globosa or malassezia furfur, the "candida albicans lipase like" homologous family consisting of various isoforms of the secretory lipase from candida albicans, and the "aspergillus lipase like" homologous family consisting mainly of hypothetical or putative lipases, mostly from aspergillus. all proteins are from organisms belonging to the subkingdom dikarya of the kingdom fungi. proteins are classified as either lipases or esterases in genbank while proteins are classified as putative or hypothetical. the only structure entry in this superfamily is from the recently resolved crystal structure of cala. based on the structure of the oxyanion hole, cala can be classified as a y-class lipase, and tyr was identified as the oxyanion hole forming amino acid. a structural comparison with other structures from the led identified a structure from the deacetylase superfamily as most closely related. a detailed structural alignment of cala with the structure of the bacillus subtilis deacetylase from the deacetylase superfamily showed a superimposition of the common α/β hydrolase fold including the catalytic triad, despite having a low overall sequence identity of only 15%. structural differences between the two structures are found in the cap and the c-terminal region. the cap region, located between β-strands and often confers substrate specificity or additional functions to the enzyme. in the case of cala, the cap region is involved in forming the tunnel like binding site for the acyl moiety. for b. subtilis deacetylase, the cap region partially shields the active site from the solvent. the cap region of cala consists of six α-helices, while the cap region of b. subtilis deacetylase consists of only four α-helices. three α-helices in both proteins are found at identical positions. cala shows an insert of three additional α-helices after the first two conserved α-helices and is missing the last α-helix of the cap present in b. subtilis deacetylase. in order to identify residues in the cap region which are conserved between the "candida antarctica lipase a like" and "deacetylases" superfamilies, a multisequence alignment of each family was performed. the two family alignments were aligned using a structural alignment of the two protein structures. the alignment demonstrated that despite the high structural similarity, there are no conserved residues in the cap region of both protein families. two hidden markov models of the three inserted α-helices of cala and the α-helices shared by both proteins were created and used to search against all other protein families of the led. no sequences with a significant similarity were found in the entire database, demonstrating that the sequence of the cap region of the "candida antarctica lipase a like" superfamily is unique. in comparison to the b. subtilis deacetylase, cala has two additional β-strands in the c-terminal region. they are positioned directly above the active site and prevent a direct access of the substrate to the active site. we assume that the β-strands and perform a lid like function for cala since movement of the two β-strands would allow substrate access to the active site of cala from a similar direction as for the b. subtilis deacetylases. a comparison of the substrate binding sites of both proteins showed that the alcohol binding site is similar in both proteins and provides ample space for alcohol moieties of substrates. therefore, both proteins are expected to accept a variety of bulky alcohols. the binding sites for the acyl moieties are highly different. cala has a long, tunnel like binding site, while the b. subtilis deacetylase has a small cavity which is part of a cleft on the protein surface. therefore, the acyl moieties of the substrates are expected to differ significantly between both enzymes. cala is expected to accept mediumt to long chain fatty acids, while the b. subtilis deacetylase is limited to short-chain acyl moieties. thus, despite the overall similarity between cala and the b. subtilis deacetylase, the acyl binding site is fundamentally different. however, the binding site of cala shows surprising similarity to another lipase, candida rugosa lipase. for crl, two different structural confirmations have been resolved, an open conformation, and a closed conformation where the lid of crl is blocking the substrate access to the active site. crl has a cap region between β-strands and consisting of four α-helices. the substrate binding site of crl consists of a long tunnel for the acyl moiety of the substrate and provides ample space for the alcohol moiety of the substrate. despite having a lower overall structure similarity to cala than the b. subtilis deacetylase, the binding sites of cala and crl are highly similar. both provide space for large, bulky alcohol moieties of the substrate and have a tunnel like binding site for the acyl moiety. both proteins posses a lid which covers the active site and prevents direct access to the substrate binding site in its closed state. the lid of crl lipase is formed by a α-helix between β-strands and and is located in the n-terminal region while the putative lid in cala is formed by the two c-terminal β-strands and. discussion the led contains annotated and systematically classified protein families of α/β hydrolases. it has been shown to be a useful tool for the systematic analysis of protein families. previous work employed the led and blast in order to identify novel enzymes belonging to the α/β hydrolase fold. a model for the prediction of protein solubility was developed and refined by performing a comprehensive analysis of the protein families of the led. a further study involved the systematic analysis of protein families of the led in regard to the distribution and conservation of functionally relevant rare codons. since the first release of the led, more than new α/β hydrolases became available and were integrated in the release. as a case study for the utility of the highly enriched and annotated database, the newly introduced superfamily of cala was analysed and compared to other protein structures in the led. the goal was to characterise the sequence and structure of cala in comparison to other α/β hydrolases despite its low sequence similarity and to understand the molecular basis of substrate recognition. while cala shows structural similarity to the deacetylase family, the substrate specificity of both enzymes differs, which is consistent with the differences observed in the substrate binding sites of both proteins. in contrast, the lipase from c. rugosa, which shows a lower overall structural similarity to cala, is remarkably similar in regard to the substrate binding site. the structural similarities and differences are in accordance with experimentally observed substrate specificities of the three enzymes. all three proteins have a spacious alcohol binding site. the b. subtilis deacetylase accepts a wide variety of bulky substrates like cephalosporin c and xylose. cala and crl also accept bulky substrates, ranging from primary alcohols to sterically hindered secondary alcohols and even tertiary alcohols. the tunnel like binding site of cala allows the enzyme to accept esters of long chain fatty acids. the similar tunnel like acyl binding site of crl also accepts fatty acids up to a chain length of. in contrast, the small acyl binding site of the b. subtilis deacetylase is unable to accept large acyl groups and is restricted towards acetyl moieties. experimentally, cala and crl have been shown to display interfacial activation. while a lid in crl has been localized and the open and closed form of crl has been crystallized, the lid function of the β-strands 9- in cala remains to be experimentally verified. however, the similarities to crl suggest a substrate access involving the movement of β-strands 9-  the analysis of the newly introduced protein family of candida antarctica lipase a demonstrates the strength of our database approach by providing a large set of protein families which share a common protein fold despite an overall low sequence similarity. by combining both, structural and sequential information of a large number of proteins a thorough analysis and classification of proteins of interest is made possible. the lipase engineering database is online accessible at http://www.led.uni-stuttgart.de. all information on families of sequence and structure data, as well as alignments, phylogenetic trees, and family-specific profiles can be accessed by manual download. 
a new superfamily for candida antarctica lipase a was introduced including the recently published crystal structure of cala. the html version of the database with new features is available at http://www.led.uni-stuttgart.de and provides sequences, structures and a set of analysis tools including phylogenetic trees and hmm profiles the lipase engineering database integrates information on sequence, structure and function of lipases, esterases and related proteins with the α/β hydrolase fold. while the α/β hydrolase fold is conserved in both proteins, the major difference is found in the cap region. a multisequence alignment of both protein families was used to create hidden markov models for the cap region of cala and showed that the cap region of cala is unique among all other proteins of the α/β hydrolase fold. by specifically comparing the substrate binding pocket of cala to other binding pockets of α/β hydrolases, the binding pocket of candida rugosa lipase was identified as being highly similar. this similarity also applied to the lid of candida rugosa lipase in comparison to the potential lid of cala. since cala has a highly divergent sequence in comparison to other α/β hydrolases, the lipase engineering database was used to classify cala in the frame of the already established classification system. the updated release was used for the evaluation of α/β hydrolases. the led serves as a valuable tool for the systematic analysis of single proteins or protein families. the new release of the lipase engineering database contains sequence entries for proteins as well as experimentally determined protein structures, including the structure of cala. sequence alignments between both proteins show a sequence similarity of only 15%.
 the lipase engineering database integrates information on sequence, structure and function of lipases, esterases and related proteins with the α/β hydrolase fold. a new superfamily for candida antarctica lipase a was introduced including the recently published crystal structure of cala. since cala has a highly divergent sequence in comparison to other α/β hydrolases, the lipase engineering database was used to classify cala in the frame of the already established classification system. this involved the comparison of cala to similar structures as well as sequence-based comparisons against the content of the led.  the new release of the lipase engineering database contains sequence entries for proteins as well as experimentally determined protein structures, including the structure of cala. in comparison to the previous release with protein and structure entries this update represents a significant increase in data volume. by comparing cala to representative structures from all superfamilies, a structure from the deacetylase superfamily was found to be most similar to the structure of cala. while the α/β hydrolase fold is conserved in both proteins, the major difference is found in the cap region. sequence alignments between both proteins show a sequence similarity of only 15%. a multisequence alignment of both protein families was used to create hidden markov models for the cap region of cala and showed that the cap region of cala is unique among all other proteins of the α/β hydrolase fold. by specifically comparing the substrate binding pocket of cala to other binding pockets of α/β hydrolases, the binding pocket of candida rugosa lipase was identified as being highly similar. this similarity also applied to the lid of candida rugosa lipase in comparison to the potential lid of cala.  the led serves as a valuable tool for the systematic analysis of single proteins or protein families. the updated release was used for the evaluation of α/β hydrolases. the html version of the database with new features is available at http://www.led.uni-stuttgart.de and provides sequences, structures and a set of analysis tools including phylogenetic trees and hmm profiles 