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 sub- families according to the PeroxiBase classiﬁcation. For each of the subfamilies the MSA contains annotation of residues conserved in the whole family and residues spe- ciﬁc 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 classi- ﬁcation allows the identiﬁcation of wrongly annotated sequences in PeroxiBase and reassignment of them to their correct sub-families. It has also improved the classi- ﬁcation of some classes of peroxidases that were diﬃcult to distinguish with classical tools. For example the classi- ﬁcation of the Vanadium peroxidase has been separated into three subcategories (bromoperoxidase, chloroper- oxidase 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). FUTURE DEVELOPMENTS The PeroxiBase is a unique, powerful and reliable data- base 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 perox- idase 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 discrimina- tors 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 subse- quent expansion of PeroxiBase will facilitate peroxidase gene-family studies. Even if the manual integration of sequences is a guar- antee of quality we need automatic methods to speed up the annotation of new sequences. Our classiﬁcation Nucleic Acids Research, 2009, Vol. 37, Database issue D265 method will help curators to rapidly integrate new perox- idases 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 scienti- ﬁc community. ACKNOWLEDGEMENTS 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. FUNDING Swiss National Science Foundation (31-068003.02 to C.D., 315200-116864 to L.C. and N.H.). Conﬂict of interest statement. None declared. REFERENCES 1. Flohe,L. and Ursini,F. (2008) Peroxidase: a term of many mean- ings. Antioxid. Redox. Signal, 10, 1485–1490. 2. Brenot,A., King,K.Y., Janowiak,B., Griﬃth,O. and Caparon,M.G. (2004) Contribution of glutathione peroxidase to the virulence of Streptococcus pyogenes. Infect. Immun., 72, 408–413. 3. Heym,B., Stavropoulos,E., Honore,N., Domenech,P., Saint- Joanis,B., Wilson,T.M., Collins,D.M., Colston,M.J. and Cole,S.T. (1997) Eﬀects of overexpression of the alkyl hydroperoxide reduc- tase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis. Infect. Immun., 65, 1395–1401. 4. Missall,T.A., Cherry-Harris,J.F. and Lodge,J.K. 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