Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

Abstract

Background: The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5.

Results: About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions.

Conclusion: Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html.

Figure 1

Phylogenetic tree of family GH5. In this circular phylogram, the branches corresponding to subfamilies 1–53 are shown in color and the subfamily numbers are indicated next to the exterior color circle. The branches corresponding to sequences not included into subfamilies are in black. A detailed version of this tree is found in Additional file 1: Figure S1.

Figure 2

Examples of modular GH5 proteins. (a) Diverse modular arrangements of putative monofunctional modular enzymes from subfamily GH5_8. (b) Same for putative bifunctional GH5 enzymes containing a subfamily GH5_8 module. (c) Other putative bifunctional enzymes containing at least a single GH5 module. (d) Selected examples of proteins containing GH5 modules having lost one or more catalytic residues. For a given protein, each GH5 module is identified by a number of fields separated by “|” indicating: (i) the organism, with 3 letters for the genre and either 5 letters for the species or full strain code; (ii) the GenBank protein accession; (iii) if attributed, the subfamily number or other information; (iv) EC numbers if available. These individual tags are analogous to what is found in Additional file 1: Figure S1. The module types and other protein segments present are: GHx_y – glycoside hydrolase family x subfamily y (pink); CEx – carbohydrate esterase module of family x (light brown); Cip21 – chitin-binding protein type 21 module with putative carbohydrate oxidative cleaving activity, formerly CBM33 (dark gray); CBMx – carbohydrate binding modules of family x (light green); FN3 – fibronectin type III modules (dark green); DOC – cellulosomal dockerin modules (light violet); EXPN – expansin modules (dark purple); signal peptides (purple); transmembrane segments (yellow); linkers (light blue); other regions (light grey).