Structural organization and a standardized nomenclature for plant endo-1,4-beta-glucanases (cellulases) of glycosyl hydrolase family 9.

Glycosyl, or glycoside, hydrolases (GHs) comprise a structurally diverse group of enzymes that hydrolyze glycosidic bonds between carbohydrates, or between carbohydrates and other noncarbohydrate moieties, and that collectively exhibit a wide range of substrate specificities. GH enzymes from across the taxonomic spectrum were originally named based on substrate specificity, the corresponding International Union of Biochemistry and Molecular Biology (IUBMB) nomenclature system (EC 3.2.1.-), and the chronological order in which they were reported. However, as growing numbers of GH proteins, and later genes, were characterized, this strategy proved to be increasingly unsatisfactory and a complementary nomenclature was developed based on predicted protein sequence (Henrissat et al., 1998). This approach provides important insights into protein structure, evolutionary relationships, and an opportunity to infer mechanistic relationships. A regularly updated database, Carbohydrate-Active Enzymes (CAZY; www.cazy.org), currently lists 108 distinct GH families, a subset of which are further affiliated with 14 clans based on the presence of defined protein folds and conserved catalytic machineries. This nomenclature initiative was developed largely in response to the rapidly growing numbers of reported microbial GHs, reflecting their numerous important industrial uses. Notable examples are endob-1,4-glucanases, or cellulases, which hydrolyze the b-1,4-glucosyl linkages of cellulose and several other plant cell wall polysaccharides, and are used in the generation of sugars from lignocellulosic biomass for ethanol production. Plant biologists are currently facing a similar nomenclature dilemma, since the sequencing of whole plant genomes has revealed many large GH families (Henrissat et al., 2001): a genomescale assessment of Arabidopsis (Arabidopsis thaliana) in CAZYidentified 393 GHs from 34 families (http://www. cazy.org/geno/3702.html), and equivalent families are emerging in many species as larger EST collections develop. GH activities from plants have long been studied in association with various aspects of growth, development, and cell wall metabolism, but plant GH enzymes have often been named with little consideration of their substrate specificity, molecular structure, or enzymatic reaction mechanism. The current explosion of interest and new research opportunities in biofuels and bioenergy crops will inevitably result in renewed interest in identifying and annotating plant GHs, through their association with lignocellulosic biomass. Thus, this is an opportune time to adopt a new, rational, well-defined nomenclature. Importantly, it is apparent that many of these plant enzymes share similarities to previously classified families of microbial GHs, which is not clear from their original designations. While the IUBMB-recommended nomenclature (www.chem.qmul.ac.uk/iubmb), based on the substrates used and the reaction catalyzed, continues to provide critical information regarding enzyme function, we propose a complementary, standardized nomenclature for plant GHs, based on the rigorous scheme for naming their microbial counterparts, which is categorized by the catalytic domain of the enzyme (Henrissat et al., 1998). We have selected plant endo-b-1,4-glucanases as a case study, since this family illustrates the problems with imprecise GH nomenclatures that have evolved over decades of research. In addition, this family of plant enzymes has a divergent subfamily structure that can usefully be incorporated into the development of a naming scheme. Early reports described the existence of plant cellulases (e.g. Hall, 1963) and cellulolytic activities have long been associated with both cell wall construction during cell expansion and the wall disassembly that accompanies processes such as fruit ripening and abscission (for review, see del Campillo, 1999; Rose and Bennett, 1999; Mølhøj et al., 2002) and cellulose biosynthesis (Nicol et al., 1998; Lane et al., 2001; Sato et al., 2001). Most of the plant ‘‘cellulases’’ studied to date are typical endoglucanases (EC 3.2.1.4) with low or no activity on crystalline cellulose, but clearly measurable activity on soluble cellulose derivatives, such as carboxymethyl cellulose, noncrystalline phosphoric acid swollen cellulose, and/or a variety of plant polysaccharide substrates, including xylans, 1,31,4-b-glucans, and glucomannans (Master et al., 2004; Yoshida and Komae, 2006; Urbanowicz et al., 2007). This is quite distinct from the case of microbial cellulases,