Published online 05 October 2008 Nucleic Acids Research, 2009, Vol. 37, Database issue D233–D238doi:10.1093/nar/gkn663

The Carbohydrate-Active EnZymes database(CAZy): an expert resource for GlycogenomicsBrandi L. Cantarel, Pedro M. Coutinho, Corinne Rancurel, Thomas Bernard,

Vincent Lombard and Bernard Henrissat*

Architecture et Fonction des Macromolecules Biologiques, UMR6098, CNRS, Universites Aix-Marseille I & II,163 Avenue de Luminy, 13288 Marseille, France

Received September 15, 2008; Accepted September 19, 2008

ABSTRACT

The Carbohydrate-Active Enzyme (CAZy) databaseis a knowledge-based resource specialized in theenzymes that build and breakdown complex carbo-hydrates and glycoconjugates. As of September2008, the database describes the present knowledgeon 113 glycoside hydrolase, 91 glycosyltransferase,19 polysaccharide lyase, 15 carbohydrate esteraseand 52 carbohydrate-binding module families.These families are created based on experimentallycharacterized proteins and are populated bysequences from public databases with significantsimilarity. Protein biochemical information is con-tinuously curated based on the available literatureand structural information. Over 6400 proteins haveassigned EC numbers and 700 proteins have a PDBstructure. The classification (i) reflects the structuralfeatures of these enzymes better than their sole sub-strate specificity, (ii) helps to reveal the evolutionaryrelationships between these enzymes and (iii) pro-vides a convenient framework to understand mech-anistic properties. This resource has been availablefor over 10 years to the scientific community, contri-buting to information dissemination and providing atransversal nomenclature to glycobiologists. Morerecently, this resource has been used to improvethe quality of functional predictions of a numbergenome projects by providing expert annotation.The CAZy resource resides at URL: http://www.cazy.org/.

INTRODUCTION

Due to the extreme variety of monosaccharide structures,to the variety intersugar linkages and to the fact that vir-tually all types of molecules can be glycosylated (fromsugars themselves, to proteins, lipids, nucleic acids,

antibiotics, etc.), the large variety of enzymes actingon these glycoconjugates, oligo- and polysaccharidesprobably constitute one of the most structurally diverseset of substrates on Earth. Collectively designated asCarbohydrate-Active enZymes (CAZymes), these enzymesbuild and breakdown complex carbohydrates and glyco-conjugates for a large body of biological roles (collectivelystudied under the term of Glycobiology). Therefore, CAZ-ymes have to perform their function usually with highspecificity. Because carbohydrate diversity (1) exceeds byfar the number of protein folds, CAZymes have evolvedfrom a limited number of progenitors by acquiring novelspecificities at substrate and product level. Such a dizzyingarray of substrates and enzymes makes CAZymes a partic-ularly challenging subject for experimental characteriza-tion and for functional annotation in genomes.Nearly 20 years ago, the first foundation for a family

classification of CAZymes was seen in an effort that clas-sified cellulases into several distinct families based onamino-acid sequence similarity (2). Soon after, thefamily classification system based on protein sequenceand structure similarities, was extended to all known gly-coside hydrolases (2–4), and subsequently extended to allCAZymes involved in the synthesis, degradation and mod-ification of glycoconjugates. The classification of CAZ-ymes has been made available on the web sinceSeptember 1998. Because based on amino-acid sequencesimilarities, these classifications correlate with enzymemechanisms and protein fold more than enzyme specifi-city. Consequently, these families are used to conserva-tively classify proteins of uncharacterized function whoseonly known feature is sequence similarity to an experimen-tally characterized enzyme, avoiding overprediction ofenzyme activities.At present, CAZy covers approximately 300 protein

families in the following classes of enzyme activities:

(1) Glycoside hydrolases (GHs), including glycosidasesand transglycosidases (3–5). These enzymes consti-tute 113 protein families that are responsible for

*To whom correspondence should be addressed. Tel: +33 4 91 82 55 87; Fax: +33 491 26 67 20; Email: Bernard.Henrissat@afmb.univ-mrs.frCorrespondence may also be addressed to Pedro M. Coutinho. Email: Pedro.Coutinho@afmb.univ-mrs.fr

� 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.

the hydrolysis and/or transglycosylation of glycosidicbonds. GH-coding genes are abundant and present inthe vast majority of genomes corresponding toalmost half—presently about 47%—of the enzymesclassified in CAZy. Because of their widespreadimportance for biotechnological and biomedical app-lications, GHs constitute so far the best biochemi-cally characterized set of enzymes present in theCAZy database.

(2) Glycosyltransferases (GTs). These are the enzymesresponsible for the biosynthesis of glycosidic bondsfrom phospho-activated sugar donors (6–8). Theyform over 90 sequence-based families and presentin virtually every single organism and representabout 41% of CAZy at present.

(3) Polysaccharide lyases (PLs) cleave the glycosidicbonds of uronic acid-containing polysaccharides bya b-elimination mechanism (6). They are presentlyfound in 19 families in CAZy (7), corresponding toonly about 1.5% of CAZy content. Many PLs havebiotechnological and biomedical applications and,despite their small overall number, they are amongthe CAZymes with the highest proportion of bio-chemically characterized examples present in thedatabase.

(4) Carbohydrate esterases (CEs). They remove ester-based modifications present in mono-, oligo- andpolysaccharides and thereby facilitate the actionof GHs on complex polysaccharides. Presentlydescribed in 15 families (7), CEs represent roughly5% of CAZy entries. As the specificity barrierbetween carbohydrate esterases and other esteraseactivities is low, it is likely that the sequence-basedclassification incorporates some enzymes that mayact on non-carbohydrate esters.

(5) Carbohydrate-binding modules (CBMs). These areautonomously folding and functioning protein frag-ments that have no enzymatic activity per se but areknown to potentiate the activity of many enzymeactivities described above by targeting to and pro-moting a prolonged interaction with the substrate.CBMs are most often associated to the other carbo-hydrate-active enzyme catalytic modules in the samepolypeptide and can target different substrate formsdepending on different structural characteristics(9,10). However, occasionally they can be presentin isolated or tandem forms not coupled with anenzyme. Roughly 7% of CAZy entries contain atleast one CBM module. CBMs are presently classi-fied in 52 families in CAZy (7).

In addition to protein families that are well curated bythe CAZy database, CAZymes are known to containdomains not acting on carbohydrates, including otherenzymes—such as proteases, myosin motors or phospha-tases, etc.—and a variety of protein–protein or protein–cell wall binding domains—cohesins, SLHs, TPR, etc.The CAZy family classification system covers all taxo-

nomic groups, and provides the ground for commonnomenclature for CAZymes across different glycobiolo-gists (11,12) generally specialized only in some specific

groups of organisms. Day-to-day inspection of newenzyme characterizations reported in the literature regu-larly led and continues to lead to the definition of newenzyme families. Significantly, the CAZy families, origin-ally created following hydrophobic cluster analysis in the1990s from very limited number of sequences available(2–6) and later complemented by BLAST- and HMMer-based sequence similarity approaches, are globally surviv-ing the challenge of time in spite of a hundred-foldincrease in the number of sequences.

DATABASE CONTENT

The CAZy database contains information from (i)sequence annotations from publicly available sources,namely the NCBI, including taxonomical, sequenceand reference information, (ii) family classification and(iii) known functional information. This data allow theexploration of an enzyme (CAZyme), all CAZymes in anorganism or a CAZy protein family. The addition of newfamily members and the incorporation of biochemicalinformation extracted from the literature are updated reg-ularly, following careful inspection. Newly released three-dimensional (3D) structures and genomes are analyzed asthey are released by public databases. Daily updatereleases from GenBank form the bulk of sequence addi-tions to the database (8) are complemented by weeklyPDB releases (13). Presently only genome releasedthrough these GenBank releases are analyzed regularly,whereas other genomes protein predictions are analyzedupon request as part of collaborative efforts (vide infra).

Another feature of CAZy is that the number of families,the family-associated information and content are con-tinuously updated. When new families are created, oldpreviously released genomes and sequence in public data-bases are reanalyzed to take the additional new family intoaccount to ensure completeness in sequence description.Internally, curators include and maintain all referencedbiochemical and other characterization data from the lit-erature and the analysis of full sets of protein sequencespresent in a single genome. Because of this continuouseffort of data addition, new families are frequentlyadded and reflect the advances in experimental character-ization of CAZymes. New families are exclusively createdbased on the availability of at least one biochemically-characterized member for which a sequence is availableand the information published in peer-reviewed scientificliterature. This sequence then serves as a seed for thefamily that is gradually extended with sequences thatshare statistically significant similarity.

Only functional assignments based on experimentaldata are included in the CAZy database by the associationof EC numbers to protein sequences. Therefore inferredfunctional assignments are not included. Experimentaldata are ideally a direct enzyme analysis, but also couldinclude indirect evidence such as gene knockout experi-ments with extensive characterization. Because there is ashortage of EC numbers, relative to the number of func-tions characterized experimentally, some incomplete ECnumbers such as 3.2.1.-, 2.4.1.-, 2.4.2.- and 2.4.99.- are

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also included in the database. In addition, as the describedfunctions in CAZy are only of enzymatic nature, addi-tional and complementary binding and inhibitory func-tions known to be associated with several CAZyproteins will be curated and explored in the near future.

SEMI-AUTOMATIC MODULAR ASSIGNMENT

Carbohydrate-active enzymes, can exhibit a modularstructure (Figure 1), where a module can be defined as astructural and functional unit (7,14). Each family in CAZyis dependent on the definition of a common segment ineach full sequence that ultimately contains the catalytic orbinding module. The definition of the limits within thesequence of the composing modules depends on availableinformation derived from a combination of differentapproaches:

(1) protein 3D structures,(2) reported deletion studies and(3) protein-sequence analysis and comparisons.

Different sequence comparison tools are used to defineenzyme families, particularly gapped BLAST (9) andHMMER (10) using hidden markov models (HMMs)made from each family. All the sequences correspondingto the catalytic and binding of carbohydrate-activeenzymes are excised from the full protein sequence andgrouped in a BLAST library. Positive hits against this‘high quality’ library, are entered into the database bytrained curators following manual check on a daily basiswith a small number of sequences with high identity(>85%) ungapped alignments to previously examinedsequences being entered automatically.

A new layer dealing with the analysis of whole proteinsets issued from genomes has been introduced recently.Modular annotation has been in fact applied to genomedata released by the NCBI, with over 750 genomes ana-lyzed. Approximately 1–3% of the proteins encoded by atypical genome correspond to CAZymes (10,11). In addi-tion to publicly released sequences, annotation of proteinsin recently sequenced genomes prior to full release areregularly performed by the CAZy team in collaborationwith scientists from all over the globe.

MANUAL FUNCTIONAL ANALYSIS

All too often, functional annotation methods employedduring whole genome annotation are erroneous and lackconsistent language (12,15). While sequence similarity togenes annotated by GO or best BLAST hits can be agood-starting point to assignment to pathways or possiblegeneral functions, such as serine/theonine kinase, manyautomatic functional assignments are unfortunatelymuch more specific. This is particularly true in the caseof CAZymes, since related families of the latter grouptogether enzymes of widely differing specificity.The CAZy database employs practices that aim to elim-

inate the problems with automatic annotation.Biochemical characterization of new proteins from theliterature is used to create new protein families, to anno-tate their referring entries and to update family descrip-tions (6). These biochemical assignments are alsoemployed to help the manual curator estimate the likelygeneral functions and add descriptions that indicate whichenzymatically characterized proteins are related to newsequences. Inclusion of reference data compiled by com-munities centered on model organisms is considered forthe future. Bibliographic references are included in CAZyby a specific layer that includes over 16 000 different bib-liographic references. These references were extractedautomatically from individual accessions using ProFal(16) and about one-third was entered manually.When functional predictions are made, they arise from

manual curation by examination of closely relatedsequences and when biochemical information is not avail-able, such as the case for many genome projects, verygeneral functional tags are used to convey general func-tions of a family. Recently, we have begun further break-ing down families into subfamilies in the hope of groupingproteins by specificity using sequence similarity. Thiswould allow us to give more insights into possible func-tions. This new classification can also give insights intoconserved active sites and active site specificity, when com-paring biochemically characterized enzymes. Currentlysubfamily assignments are available publicly only forGH13 (14), GH1, GH2 and GH5 (released with this pub-lication). This effort will be continued in the future withmany more subfamilies being incorporated into the CAZyknowledge base in the future. Subfamilies identify sub-groups of sequences that are more homogeneous in theirfunctional properties. Most identified subfamilies aremonospecific. If polyspecific, the functional variability islow and typically limited to two or three EC activities.There, often the known subfamily functions often sharea substrate or product. Furthermore, rational enzymeengineering may be used to switch the functions for severalcases (data not shown). Subfamilies also open the door forfurther enzymatic characterization—a few subfamilies asstill no known activity—or for the identification of mean-ingful targets for structural characterization.

LARGE-SCALE ANALYSIS AND COLLABORATION

Internal CAZy tools, such as our semi-automatic modularassignment presently allow the analysis of a larger number

(a)

(b)

(c)

(d)

(e)

(f)

Figure 1. Examples of modular carbohydrate-active enzymes. (a)Cellobiohydrolase I from Hypocrea jecorina (SP P00725); (b) alginatelyase from Sphingomonas sp. A1 (GB BAB03312.1); (c) xylanase fromCellulomonas fimi (GB CAA54145.1); (d) xylanase D/lichenasefrom Ruminococcus flavefaciens (GB CAB51934.1); (e) chitin synthasefrom Emericella nidulans (GB BAA21714.1); (f) cyclicb-1-3-glucansynthase from Bradyrhizobium japonicum (GB AAC62210.1).

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of sequences than a few years ago, making it possible toperform large-scale analyses, such as the annotation ofCAZyme systems in genomes and metagenomic investiga-tions of the breakdown of complex carbohydrates. A typi-cal genome analysis begins with the assignment of proteinmodels to one or several CAZy families (depending on thenumber of CAZy modules present within the sequence).This family assignment is then followed by the predictionof general functional classes using a manual examinationof alignments to closely related sequences, taking care toidentify the retention of active-site residues. Once agenome is categorized by family and functional classes,gene content analysis is utilized to give insights into hownewly sequenced organisms might be similar or differentfrom closely related species. Differences in genome con-tent, i.e. relative family size, might reflect the relativediversity or complexity of the inherent biological processes(17) and therefore, the biology of the compared species.For example, differences suggesting a more pronouncedpectin metabolism in ‘dicot’ Arabidopsis versus ‘monocot’rice have been noted (17) as well as expected differences incell-wall metabolism between short-lived annualArabidopsis versus long-lived poplar tree have been sug-gested (18). With the advent of a variety of post-genomictechniques, a new vision of the CAZymes as significant

components of carbohydrate-based systems now emerges.Examples include: N- and O-glycosylation of proteins,starch metabolism, biosynthesis of the cell-wall and itssubcomponents. Geisler-Lee et al. (19) have combinedbioinformatics and transcriptome analysis of variouspoplar and Arabidopsis tissues and organs and haveshown that CAZyme transcripts are particularly abundantin wood tissues.

NEW FEATURES

In addition to a website facelift, the new CAZy websitecomes with a host of new features. Primarily, we are nowoffering users the ability to search the CAZy site for infor-mation by GenBank protein accession number, family ororganism rather than navigate long static pages as prior to12/31/2008 (Figure 2). To the new site we are also includ-ing, pages to describe new releases, new genomes andother new features. In addition, tools developed in thelab are available for interactive use.

FUTURE TRENDS

The CAZy database is a fluid database always changingand growing as additional data becomes available.

A

B

Figure 2. (A) Once a search is performed, such as for a protein accession (P00275), the resulting page indicates the modular families that composethat protein. (B) Upon clicking the resulting links provided in A, users are directed to a page about the family and gives a listing of all annotatedmembers.

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In the last 2 years, the number of sequences in CAZY hasnearly doubled and the number of available genomes isover 750. We believe this trend will continue in the comingyears. Unfortunately, while sequencing is forever morerapid, progress in structural information and biochemicalcharacterization is much slower. The number of biochem-ical data has grown only by 8% over the last 2 years(Figure 3). This means that the gap is widening betweenavailable sequences and biochemically characterizedenzymes, making better methods for high-throughput bio-chemical characterization advantageous.

As started previously, we are actively pursuing the clas-sification of subfamilies within each family. This furtherlevel of classification is important for instance to identifykey residues or motifs important to define specificity.Finally, we hope to offer soon a page to submit sequencesfor a sequence similarity search and keyword search onour website.

AVAILABILITY ON THE WEB

The CAZy database is available at www.cazy.org.Information about selected families is available through

the website and at www.cazypedia.org. Software fromthe group is available at www.cazy.org/tools.

FUNDING

The authors wish to thank the Departement des Sciencesde la Vie of CNRS for a 2-year funding grant to B.L.C.and Novozymes for a contract supporting V.L.

Conflict of interest statement. P.M.C. is affiliated toUniversite de Provence (Aix-Marseille I) and B.H. andC.R. are members of CNRS.

REFERENCES

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14. Stam,M.R., Danchin,E.G., Rancurel,C., Coutinho,P.M. andHenrissat,B. (2006) Dividing the large glycoside hydrolasefamily 13 into subfamilies: towards improved functionalannotations of alpha-amylase-related proteins. Protein Eng. Des.Sel., 19, 555–562.

15. Gilks,W.R., Audit,B., De Angelis,D., Tsoka,S. and Ouzounis,C.A.(2002) Modeling the percolation of annotation errors in a databaseof protein sequences. Bioinformatics (Oxford, England), 18,1641–1649.

16. Couto,F.M., Silva,J.M. and Coutinho,P.M. (2003) ProFAL:PROtein Functional Annotation through Literature. In Pimentel,E.,Brisaboa,N.R. and Gomez, J. (eds), In Proceedings of the 8th

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Figure 3. The number of protein containing CAZymodules were noted inDecember of the years 1999–2007. Within this set (Open circle), thenumber of enzymatically characterized proteins (triangle) and thosewith solved structures (open diamond) were also counted. In December2007, <10% of proteins in CAZy were characterized enzymatically and<1% had a solved structure. In 8 years, the number of sequences hasincreased 14-fold, while the number of enzymatic and structural charac-terization has mearly doubled. Therefore, the porportion of proteins withfunctional and stuctural information is decreasing rapidly unless highthroughput functional efforts are made in this category of enzymes.

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Conference on Software Engineering and Databases, Alicante, Spain,pp. 747–756.

17. Yokoyama,R., Rose,J.K. and Nishitani,K. (2004) A surprisingdiversity and abundance of xyloglucan endotransglucosylase/hydrolases in rice. Classification and expression analysis.Plant Physiol., 134, 1088–1099.

18. Tuskan,G.A., Difazio,S., Jansson,S., Bohlmann,J., Grigoriev,I.,Hellsten,U., Putnam,N., Ralph,S., Rombauts,S., Salamov,A.

et al. (2006) The genome of black cottonwood,Populus trichocarpa (Torr. & Gray). Science (New York, NY),313, 1596–1604.

19. Geisler-Lee,J., Geisler,M., Coutinho,P.M., Segerman,B.,Nishikubo,N., Takahashi,J., Aspeborg,H., Djerbi,S., Master,E.,Andersson-Gunneras,S. et al. (2006) Poplar carbohydrate-activeenzymes. Gene identification and expression analyses.Plant Physiol., 140, 946–962.

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