Nucleic Acids Research Advance Access published online on October 23, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn680
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
PeroxiBase: a database with new tools for peroxidase family classification
Dominique Koua1,2,
Lorenzo Cerutti1,
Laurent Falquet3,
Christian J. A. Sigrist1,
Grégory Theiler2,
Nicolas Hulo1 and
Christophe Dunand2,*
1Swiss Institute of Bioinformatics, Swiss-Prot Group, CMU, 1 rue Michel Servet, 2Laboratory of Plant Physiology, University of Geneva, Quai Ernest-Ansermet 30, CH-1211 Geneva 4 and 3Swiss Institute of Bioinformatics, EMBnet Group, Quartier Sorge - Bâtiment Génopode, CH-1015 Lausanne, Switzerland
*To whom correspondence should be addressed. Tel: 0033 562 193 557; Fax: 0033 562 193 502; Email: dunand{at}scsv.ups-tlse.fr Present address: Christophe Dunand, SCSV-UMR5546 CNRS/UPS, 24 Chemin de Borderouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France
Received August 15, 2008. Revised September 19, 2008. Accepted September 23, 2008.
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ABSTRACT
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Peroxidases (EC 1.11.1.x), which are encoded by small or large
multigenic families, are involved in several important physiological
and developmental processes. They use various peroxides as electron
acceptors to catalyse a number of oxidative reactions and are
present in almost all living organisms. We have created a peroxidase
database (
http://peroxibase.isb-sib.ch) that contains all identified
peroxidase-encoding sequences (about 6000 sequences in 940 organisms).
They are distributed between 11 superfamilies and about 60 subfamilies.
All the sequences have been individually annotated and checked.
PeroxiBase can be consulted using six major interlink sections
‘Classes’, ‘Organisms’, ‘Cellular
localisations’, ‘Inducers’, ‘Repressors’
and ‘Tissue types’. General documentation on peroxidases
and PeroxiBase is accessible in the ‘Documents’
section containing ‘Introduction’, ‘Class
description’, ‘Publications’ and ‘Links’.
In addition to the database, we have developed a tool to classify
peroxidases based on the PROSITE profile methodology. To improve
their specificity and to prevent overlaps between closely related
subfamilies the profiles were built using a new strategy based
on the silencing of residues. This new profile construction
method and its discriminatory capacity have been tested and
validated using the different peroxidase families and subfamilies
present in the database. The peroxidase classification tool
called PeroxiScan is accessible at the following address:
http://peroxibase.isb-sib.ch/peroxiscan.php.
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INTRODUCTION
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Peroxidases are enzymes that use various peroxides (ROOH) as
electron acceptors to catalyze a number of oxidative reactions.
These peroxidases can be haem and non-haem proteins. They are
extremely widespread and present in all living organisms. In
mammals, they are implicated in biological processes as various
as immune system or hormone regulation. In plants, they are
involved in auxin metabolism, lignin and suberin formation,
cross-linking of cell wall components, defense against pathogens
or cell elongation. Humans contain more than 30 peroxidases
whereas
Arabidopsis thaliana has about 130 peroxidases that
are grouped in 13 different families and nine subfamilies. There
has been increased interest over the last few years in the role
that mammalian haem peroxidase enzymes may play in both disease
prevention and human pathologies. In general, haem peroxidases
tend to promote rather than inhibit oxidative damage. Some mammalian
haem peroxidases use H
2O
2 to generate more aggressive oxidants
to fight intruding micro-organisms (
1). Peroxidase families
from prokaryotic organisms, protists and fungi have been shown
to promote virulence (
2–5).
At the biochemical level, peroxidases can be found in the same enzyme sub-subclass E.C.1.11.1.x, donor:hydrogen-peroxide oxidoreductase (6). Currently, 15 different EC numbers have been ascribed to peroxidase: from EC 1.11.1.1
[EC]
to EC 1.11.1.16
[EC]
(EC 1.11.1.4
[EC]
was removed) (7). Other peroxidase families with dual enzymatic domains were classified with the following numbers: EC 1.13. 11.44, EC 1.14.99.1
[EC]
, EC 1.6.3.1
[EC]
and EC 4.1.1.44
[EC]
(7). The two independent EC numbers (1.11.1.9
[EC]
and 1.11.1.12
[EC]
) both correspond to glutathione peroxidase and are based on the electron acceptor (hydrogen peroxide or lipid peroxide, respectively). Two particular cases are also observed for numbers EC 1.11.1.2
[EC]
(NADPH peroxidase) and 1.11.1.3
[EC]
(fatty acid peroxidase) and no known peroxidase sequence has been assigned to NADPH peroxidase. Peroxidasins, peroxinectins, other non-animal peroxidases, Dyp-type peroxidases, hybrid ascorbate-cytochrome C peroxidases and other Class II peroxidases do not possess their own EC number and can only be classified in EC 1.11.1.7
[EC]
.
At the sequence level, most haem peroxidases belong to two large families, one mainly found in plants and also in bacteria and fungi (7, 8), and a second found mostly in animals (but also occasionally in some fungal and bacterial species) (9, 10). These two independent groups, though possessing weak sequence homology, can still be identified with a common signature (see InterPro entry IPR010255). In addition to these two large superfamilies, four smaller protein families are indexed as capable of reducing peroxide molecules with the help of haem. Catalases (Kat), which can also oxidize hydrogen peroxide (unique feature); Di-haem cytochrome C peroxidases (DiHCcP); Dyp-type peroxidases (DypPrx); and haem Haloperoxidases (HalPrx). These families display no sequence homology between each other.
Non-haem peroxidases are not evolutionarily linked and form five independent families. The largest one is the thiol peroxidase, which currently contains more than 1000 members grouped in two different subfamilies (Glutathione peroxidases and Peroxiredoxines). Alkylhydroperoxidase, non-haem haloperoxidase, manganese catalase and NADH peroxidase are the remaining other four non haem peroxidase families.
According to the phylogenetic trees these 11 major groups can be subdivided in 60 subfamilies (Figure 1). These subdivisions based on evolution describe quite well the variety of peroxidase functions and can thus be used to predict the function of newly characterized proteins.
Due to the high diversity of peroxidase functions and increased
interest of the medical research in pathologies related to the
role of peroxidases there is an urgent need to federate and
organize data on peroxidases. The goal of our database is to
centralize most sequences that belong to peroxidase superfamilies,
to follow the evolution of peroxidase among living organism
and to compile the information concerning putative functions
and transcriptional regulation. Currently, PeroxiBase is a unique
repository exclusively dedicated to peroxidase families and
superfamilies from both Eukaryotes and Prokaryotes. It includes
6000 peroxidases encoding sequences from 940 organisms, and
each sequence is individually annotated. We have also developed
a new tool to facilitate the classification of new peroxidase
members.
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DATABASE INTERFACE ORGANIZATION
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The PeroxiBase toolbar is divided into eight sections (
Figure 2).
The ‘Documents’ tab gives access to general information:
‘Introduction’, ‘Class description’,
‘Publications’ and ‘Links’. Several
useful tools are available (‘Tools’) to classify
and analyse peroxidases: ‘Search’ permits complex
text queries on the database, ‘Blast’ allows a comparison
between a query sequence and the peroxidases stored in PeroxiBase
and, ‘FingerPrintscan’ and ‘PeroxiScan’
help classify a query sequence in the right group. The six following
sections named ‘Classes’, ‘Organisms’,
‘Cellular localisations’, ‘Inducers’,
‘Repressors’ and ‘Tissue types’ permit
the user to navigate within PeroxiBase using the specified criteria.
Individual data sheets have been largely redesigned since the
previous PeroxiBase publication (
Figure 2).
Last sequence changes, Reviewer and
Last annotation changes fields exhibit the date
of first entry (or of last sequence modification) with name
of the contributor; the name of the curator who checked the
entry, and the date of the last modification in any sections
with name of the contributor, respectively. In an attempt to
set up a unified nomenclature (
Name field), we introduced a
simple nomenclature based on species and class acronyms. The
various original appellations have been conserved as synonyms
in PeroxiBase.
Class field refers to the class the peroxidase
belongs based on the new PeroxiScan tool.
Cellular localisation, Tissue type, Inducer and
Repressor fields present data concerning
the gene and protein expressions. These fields use fixed terms.
Best BLASTp hits field reports the five closest hits to this
entry obtained from daily updated BLAST searches.
Protein ref, DNA ref, mRNA ref and
Cluster/prediction ref fields refer to
hyperlinks protein, DNA, mRNA sequences and cluster respectively.
PeroxiBase entries are cross-referenced in UniProtKB (SwissProt/TREMBL).
The data are stored in a MySQL relational database and the web
interface is made of PHP and CGI/Perl scripts.
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Figure 2. Screenshot of one entry and description of the toolbar. The toolbar includes various sections. ‘Documents’ contains ‘Introduction’, ‘Class description’, ‘Publications’ (related to PeroxiBase) and ‘Links’ (specific and general databases). ‘Tools’ menu contains the following sections: ‘Search’ (multi criteria), ‘Blast’, ‘PeroxiScan’ and ‘FingerPrintscan’.
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DATA ACQUISITION AND INTEGRATION
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The automatic annotation of the complete genomes of numerous
organisms and the automatic clustering and assembling of EST
sequences led to the identification of numerous sequences coding
for different peroxidase families and superfamilies. However,
the automatic processing of the sequences is known to be of
poor quality or not as specific as expected. Using the highly
conserved motifs of each peroxidase class, manual annotation
and editing can clearly identify the correct sequences even
in low-quality sequences. In order to increase data reliability,
each new entry is individually controlled by a database curator.
Each cross-reference is verified by the reviewer. The quality
of the sequence is also examined by performing a sequence alignment
with the other homologous sequences.
Thank to the continuous release of numerous genome sequencing projects (525 in March 2007 and 843 in August 2008 according to the Genomes OnLine Database (11)) and EST libraries, existing entries can be updated and, as more annotated sequences are integrated, the organism coverage is also increased. Existing entries are frequently verified and updated if any changes have occurred.
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NEW CLASSIFICATION TOOLS FOR PEROXIDASES
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To facilitate the classification of newly sequenced peroxidase
proteins, we have developed a tool, based on PROSITE profile
methodology that takes advantage of the manually curated hierarchical
classification of PeroxiBase. One major problem with subfamily
classification is the difficulty in separating proteins due
to their high degree of similarity at the sequence level. The
main principle of our new approach is to build a PROSITE profile
on the whole conserved region of each subfamily, but to make
the profile more specific, residues that are conserved in the
whole family are lightened and residues specific to each subfamily
are emphasized. We started by merging all families that overlap
to construct general alignments. In these alignments, we specifically
tag well-conserved residues. The family alignments are then
simply split (without modifying the alignment of residues) in
several sub-alignments according to our subfamily classification.
Each subfamily alignment now contains an annotation line where
residues conserved in the whole family and residues specific
to the subfamily are tagged. This annotation line is then used
by our profile construction program to down weigh family-conserved
columns and over weight subfamily-specific ones (see
http://www.expasy.org/tools/subprofiler/subprofiler_help.html for more details).
We first built profiles or used existing PROSITE profiles for the 11 major families that do not overlap. We used these profiles to build multiple sequence alignments (MSA) and integrate the PeroxiBase classification into the MSA. These 11 families were then split into 60 subfamilies according to the PeroxiBase classification. For each of the subfamilies the MSA contains annotation of residues conserved in the whole family and residues specific to the subfamily. This information was used to build 60 sub-profiles specific to each subfamily, which cover all the diversity of peroxidases. During the scanning process the various sub-profiles are in competition and only the best score is reported as is done for overlapping profiles in the PROSITE database (12). This sub-profile classification allows the identification of wrongly annotated sequences in PeroxiBase and reassignment of them to their correct sub-families. It has also improved the classification of some classes of peroxidases that were difficult to distinguish with classical tools. For example the classification of the Vanadium peroxidase has been separated into three subcategories (bromoperoxidase, chloroperoxidase and iodoperoxidase). Each profile is associated to a specific function or to a biological process in order to facilitate functional classification of newly discovered proteins. New sequences can be scanned against the subfamily profile peroxidases at the following address: http://peroxibase.isb-sib.ch/peroxiscan.php (Figure 3). Fine descriptions of the matching residues as well as matching scores can be obtained from a direct submission through MyHits web site (http://.myhits.isb-sib.ch) (13).
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Figure 3. The new PeroxiScan interface and result. PeroxiScan tool enables the identification of a given peroxidase sequence. PeroxiScan can be performed directly from one entry or independently from the ‘Tools’ section for an unknown sequence. Fine descriptions of the matching scores are available from a direct submission through MyHits web site.
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FUTURE DEVELOPMENTS
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The PeroxiBase is a unique, powerful and reliable database dedicated
to a large superfamily composed of several families (multigenic
or not) and present in all kingdoms. The database currently
contains over 6000 complete or partial peroxidase-encoding sequences
distributed among 60 different protein classes. The number of
peroxidase families should not undergo major changes in the
future. We expect only minor modifications in the sub-classification
of a few classes due to better coverage and to the biochemical
characterization of the enzymes. Profiles will be updated continuously
to account for such modifications, thus maintaining high quality
discriminators to pursue our effort in data mining of non-annotated
sequences.
Even with the large extension of the database (from 4700 in March 2007 (14) to 6026 in August 2008), it is still mainly composed of sequences originated from Viridiplantae (68%). The next step forward is to extend the coverage and to increase the number of sequences from exotic and poorly represented organisms. As the number of new sequences increases rapidly, the subsequent expansion of PeroxiBase will facilitate peroxidase gene-family studies.
Even if the manual integration of sequences is a guarantee of quality we need automatic methods to speed up the annotation of new sequences. Our classification method will help curators to rapidly integrate new peroxidases and assign them to the correct sub-families.
To make the PeroxiBase more user-friendly to anyone who would like to add new entries or to modify present entries, a Wiki page is in development. It will surely create more collaborative interactions for the peroxidase scientific community.
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FUNDING
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Swiss National Science Foundation (31-068003.02 to C.D., 315200-116864
to L.C. and N.H.).
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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We thank Filippo Passardi, Nenad Bakalovic and Vassilios Ioannidis
for their efforts in the development of the PeroxiBase database,
as well as the Swiss Institute of Bioinformatics for web hosting.
We are also indebted to Amos Bairoch and his team for cross-referencing
PeroxiBase entries in UniProt Knowledgebase, and Tania Lima
for critical reading of the article.
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REFERENCES
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ABSTRACT
INTRODUCTION