The Lipase Engineering Database: a navigation and analysis tool for protein families.

Abstract

The Lipase Engineering Database (LED) (http://www.led.uni-stuttgart.de) integrates information on sequence, structure, and function of lipases, esterases, and related proteins. Sequence data on 806 protein entries are assigned to 38 homologous families, which are grouped into 16 superfamilies with no global sequence similarity between each other. For each family, multisequence alignments are provided with functionally relevant residues annotated. Pre-calculated phylogenetic trees allow navigation inside superfamilies. Experimental structures of 45 proteins are superposed and consistently annotated. The LED has been applied to systematically analyze sequence-structure-function relationships of this vast and diverse enzyme class. It is a useful tool to identify functionally relevant residues apart from the active site residues, and to design mutants with desired substrate specificity.

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The Lipase Engineering Database: a navigation
and analysis tool for protein families
Markus Fischer and Ju ¨rgen Pleiss*
Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany
Received August 14, 2002; Revised and Accepted August 29, 2002
ABSTRACT
The Lipase Engineering Database (LED) (http://
www.led.uni-stuttgart.de) integrates information on
sequence,structure,and
esterases, and related proteins. Sequence data on
806 protein entries are assigned to 38 homologous
families, which are grouped into 16 superfamilies
with no global sequence similarity between each
other. For each family, multisequence alignments
are provided with functionally relevant residues
annotated. Pre-calculated phylogenetic trees allow
navigation inside superfamilies. Experimental struc-
tures of 45 proteins are superposed and consistently
annotated. The LED has been applied to system-
atically analyze sequence–structure–function rela-
tionships of this vast and diverse enzyme class. It is
ausefultooltoidentify
residues apart from the active site residues, and to
design mutants with desired substrate specificity.
functionoflipases,
functionallyrelevant
INTRODUCTION
Lipases (triacylglycerol hydrolases E.C. 3.1.1.3) are ubiquitous
enzymes which play an important role in lipid metabolism, as
they catalyze hydrolysis and synthesis of triglycerides and other
water insoluble esters. Microbial lipases are widely used
enzymes in biotransformation due to their high stability,
activity, regio- and stereoselectivity (1,2). Although lipases
belong to many different protein families without sequence
similarity, they have the same architecture, the a/b-hydrolase
fold (3) and a conserved active site signature, the GxSxG-
motif (4). They all share the same catalytic machinery
consisting of a catalytic triad (serine–histidine–aspartic or
glutamic acid) and the oxyanion hole, formed by the backbone
amides of two conserved residues. Experimental structure
determination of free enzymes and complexes with substrate-
analogous inhibitors (5–7) revealed insights into the structural
determinants of enzymatic function, substrate specificity and
selectivity (8,9). Otherenzymeswiththesame fold and catalytic
machinery are esterases (E.C. 3.1.1) like acetylcholinesterases
(E.C. 3.1.1.7), cutinases (E.C. 3.1.1.74), carboxylesterases
(E.C. 3.1.1.1), arylesterases (E.C. 3.1.1.2), phospholipases A1
(E.C. 3.1.1.32), cholinesterases (E.C. 3.1.1.8), and juvenile-
hormone esterases (E.C. 3.1.1.59), but also thioesterases
(E.C. 3.1.2.14) and non-heme peroxidases (E.C. 1.11.1.10).
The lipase family also includes proteins which are homologous
to lipases or esterases, but have no enzymatic function like
gliotactin, glutactin, neurotactin, neuroligin and thyroglobulin.
TheLipaseEngineeringDatabase(LED)hasbeendesignedto
serve as a navigation tool for systematic analysis of the
relationship of sequence, structure, and function of this rapidly
growing, highly diverse protein class, and for the design of
variants with optimized properties. The LED integrates
information on sequence and structure. Proteins are assigned
to homologous families and superfamilies based on sequence
similarity. To study sequence variation inside and between
families, sequence alignments are provided for each family.
Functionally relevant residues which are involved in substrate
bindingorcatalysis areannotated.Tocompare thebinding sites,
experimentally determined structures are superposed and
annotated. A preliminary version of the database (10), although
based on manually created HTML files and restricted to a
limitednumberofentries,wasusefulforadeeper understanding
of this protein class (10,11). The new version is based on an
extensible data model and a relational databasewhich integrates
sequence, structure, and annotation information available in the
public databases GenBank (12) and PDB (13).
CONSTRUCTION
Based on the classification of a preliminary release of the
LED (10) representative sequences of 37 homologous families
were selected. Theywere used to perform BLAST searches (14)
in the non-redundant sequence database at GenBank with a
cutoff of E¼10?10. An automated retrieval system extracted
each resulting hit, parsed information on sequence, source
organism, protein features, and descriptions, and loaded it into a
relational database. From this information, a pre-classification
into protein families was achieved. In a second round, protein
entries were evaluated by multisequence alignments. All
sequences with an overall sequence identity of more than
95% were assigned to a single protein entry. For each protein
entry the longest sequence was selected as a reference sequence
for multisequence alignments. For GenBank entries referring to
a PDB entry, monomers were extracted from the ExPDB
*To whom correspondence should be addressed: Tel: þ49 711 6853191; Fax: þ49 711 6853196; Email: juergen.pleiss@po.uni-stuttgart.de
# 2003 Oxford University Press Nucleic Acids Research, 2003, Vol. 31, No. 1
DOI: 10.1093/nar/gkg015
319–321

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database (15), superposed (11), and secondary structure
information was created by DSSP (16).
After data extraction the sequences were analyzed. All
sequences with high similarity were assigned to a single
homologous family. Homologous families with low, but
significant sequence similarity between each other were
grouped into a single superfamily. Superfamilies have no
significant sequence similarity between each other. Analysis
occurred in three subsequent steps: (1) Sequences which were
incorrectly assigned to protein entries were reassigned.
(2) Based on multisequence alignments and phylogenetic trees
of superfamilies, classification of homologous families and
assignment of proteins to families was refined. If necessary,
proteins were reclassified or new homologous families were
created. (3) Finally, extracted annotation information was
validated by analyzing conservation inside homologous
families and superfamilies, and by comparing to structure
information if available. Missing annotation was completed by
transferring information among conserved residues in multi-
sequence alignments. Contradictory annotation was removed.
This protocol resulted in a three-level hierarchy (superfamily,
homologous family, sequence) with a most complete and
consistent annotation of functionally relevant residues.
PROTEIN SEQUENCES
The LED contains 1367 sequences for 806 protein entries with
29% being putative proteins. For 45 protein entries, 150
structure data sets from PDB are available with a total number
of 198 protein chains. Proteins were assigned to either of two
classes, the GX and the GGGX class, according to sequence
and structure of the oxyanion hole (10). The GX class consists
of 11 superfamilies and 22 homologous families, which
contain 376 protein entries with 600 sequences and 125 chains
of known structure. This class comprises mainly bacterial and
fungal lipases, eukaryotic lipases (hepatic, lipoprotein, pan-
creatic, gastric, and lysosomal acid lipases), cutinases,
phospholipases and non-heme peroxidases. The GGGX class
consists of 5 superfamilies and 16 homologous families
including 430 protein entries with 767 sequences and 73
chains of known structure. It comprises bacterial esterases,
a-esterases, eukaryotic carboxylesterases, bile-salt activated
lipases, juvenile hormone esterases, hormone sensitive lipases,
acetylcholinesterases, and thioesterases, as well as gliotactin,
glutactin, neurotactin, neuroligin, and thyroglobulin.
ANNOTATED MULTISEQUENCE ALIGNMENTS
For each superfamily and each homologous family, multi-
sequence alignments were generated using CLUSTALW (17)
for one representative sequence per protein. For protein
structures, all sequence entries were included and displayed
with aligned secondary structure information. Proteins were
labeled by the accession code of the source database; each label
was linked to the original GenBank entry. Annotation
information of individual residues (catalytic triad, oxyanion
hole, anchor residue, disulfide bridges, scissile fatty acid
binding site, alcohol binding site, mutations, glycosylation
sites, signal, propeptide, ER retention signal, lid) are visualized
by color-coding of the multisequence alignment. Upon
selection of a colored residue, information on its position and
all annotation information concerning this residue is displayed.
Secondary structure information was calculated using DSSP
and aligned to the corresponding protein sequence.
PHYLOGENETIC ANALYSIS
Phylogenetic trees for superfamilies and homologous families
were calculated from the multisequence alignments using
TREE-PUZZLE (18), visualized using PHYLODENDRON,
and manually edited. The nodes representing the reference
sequences are labeled and linked to the corresponding
GenBank entry. A statistical significance is given for each
branching by a bootstrap analysis.
The phylogenetic tree organizes the proteins by sequence
similarity. However, this does not necessarily coincide with
relationships between the source organisms. On one hand,
sequences of a homologous family frequently come from very
different organisms, like the homologous family for lipase 2
from Moraxella which comprises proteins from bacteria, fungi
and eukaryotes. On the other hand, lipase and esterase
sequences from the same genome are widely spread over
the phylogenetic tree, as for Drosophila melanogaster, where
85 lipases and lipase-related proteins have been assigned to
5 different superfamilies and 14 homologous families.
ACCESSIBILITY
Information on superfamilies and homologous families,
and tables of the protein entries are accessible at http://
www.led.uni-stuttgart.de. The HTML pages can be accessed
by any JavaScript capable WWW browser. Protein tables
provide information on the protein name, the source organism,
accession codes and links to the GenBank entries, short
descriptions for the sequence entries, and links to fully
annotated multisequence alignments, phylogenetic trees, and
superposed protein structures if available. In addition, BLAST
searches can be performed against all sequences represented by
the database. The most similar hits and their corresponding
homologous family and superfamily are listed in a HTML
document and linked to the respective LED protein entries.
APPLICATIONS
A systematic analysis of sequence and structure of lipases has
led to a deeper insight into the relation between sequence,
structure, and function. From sequence alignment and structure
superposition it was observed that the oxyanion hole is highly
conserved inside the GX and the GGGX class (10). It could be
shown recently that this classification has important conse-
quences for activity. Numerous esterases and lipases of the
GGGX class were screened for activity towards esters of
tertiary alcohols, and most of them showed activity and even
enantioselectivity (19). In contrast, none of the GX class
enzymes was active towards this class of bulky substrates. This
local pattern has a high predictive value and can be used for
distinguishing active from inactive enzymes.
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While the residue X in the GGGX class is mostly a
hydrophobic residue, it is either hydrophobic or hydrophilic in
the GX type (10). However, it is highly conserved inside
homologous families and superfamilies. By comparing the
orientation of the side chain it was observed that it interacts
with a highly conserved ‘anchor residue’ outside the active
site. Although it does not contact the substrate, mutation
studies (20,21) demonstrated that this residue is part of a
functionally relevant network which stabilizes the local
geometry of the oxyanion hole. The residues of this network
can be identified by systematic comparisons inside protein
families and across family borders.
Similarly, results from mutation analysis of substrate
binding can be interpreted by annotating all fatty acid binding
residues (11). Replacement of F95 which is part of the scissile
fatty acid binding site of Rhizopus lipase by the more bulky
and hydrophilic tyrosine decreases binding of long chain fatty
acids (22). More drastically, the binding tunnel of Candida
rugosa lipase can be completely blocked for fatty acids longer
than C6 by replacing an appropriately positioned P246 by
phenylalanine (23).
A careful and comprehensive analysis of sequence and
structure similarities between lipases and esterases also allows
identification of conserved sequence patterns which are specific
for superfamilies, and thus the design offamily-specific primers
for screening DNA for new members of a superfamily.
The LED system was developed and applied for the class of
lipase-related enzymes. However, it has been designed in such
a way that it can be easily adapted to investigate other protein
families with an even larger number of members. To the expert
scientist working with a large protein class, it may serve as a
useful tool to organize information, analyze sequence–
structure–function relationships, and navigate among highly
diverse protein families.
ACKNOWLEDGEMENT
This work was supported by the German Federal Ministry of
Education and Research (project PTJ 31/0312702).
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