Tracing determinants of dual substrate specificity in glycoside hydrolase family 5

Abstract

Enzymes are traditionally viewed as having exquisite substrate specificity; however, recent evidence supports the notion that many enzymes have evolved activities against a range of substrates. The diversity of activities across glycoside hydrolase family 5 (GH5) suggests that this family of enzymes may contain numerous members with activities on multiple substrates. In this study, we combined structure- and sequence-based phylogenetic analysis with biochemical characterization to survey the prevalence of dual specificity for glucan- and mannan-based substrates in the GH5 family. Examination of amino acid profile differences between the subfamilies led to the identification and subsequent experimental confirmation of an active site motif indicative of dual specificity. The motif enabled us to successfully discover several new dually specific members of GH5, and this pattern is present in over 70 other enzymes, strongly suggesting that dual endoglucanase-mannanase activity is widespread in this family. In addition, reinstatement of the conserved motif in a wild type member of GH5 enhanced its catalytic efficiency on glucan and mannan substrates by 175 and 1,600%, respectively. Phylogenetic examination of other GH families further indicates that the prevalence of enzyme multispecificity in GHs may be greater than has been experimentally characterized. Single domain multispecific GHs may be exploited for developing improved enzyme cocktails or facile engineering of microbial hosts for consolidated bioprocessing of lignocellulose.

FIGURE 1.

Phylogeny of glycoside hydrolase family 5. A phylogenetic tree of the GH5 family constructed from a structure-based sequence alignment is shown. Experimental characterizations of function from the CAZy database are depicted in the outer rings for endoglucanases (EC 3.2.1.4; orange), mannanases (3.2.1.78; blue), 1,3-β-glucosidases (3.2.1.58; purple), and other functions (red). Genes with structures are represented by black boxes. Tree branches of genes predicted in this work to have dual endoglucanase and mannanase activities are colored pink. Subfamilies A1–A12 are labeled. (Created with the interactive Tree of Life (42).)

FIGURE 2.

Determination of positions affecting specificity in glycoside hydrolase family 5. a, sequence profiles of the active site positions in the GH5 subfamily containing Cel5A_Tma (A4), of two predominantly mannanase subfamilies (A7 and A8), and of three predominantly endoglucanase subfamilies (A1, A2, and A5/6) (created with WebLogo (43)). b, experimental measurements of the relative specific endoglucanase and mannanase activities of alanine mutants at positions in the Cel5A_Tma active site. Residues that are variable on average between A4 and the other subfamilies are labeled with a green circle (see “Experimental Procedures” for details). Data in panel b are means from three independent experiments; error bars show S.D.

FIGURE 3.

Characterization of additional GH5 A4 subfamily genes for dual specificity on glucan and mannan. a, experimental characterization of the endoglucanase (orange) and mannanase (blue) activities of Cel5A_Tma and 10 other genes from GH5 subfamily A4. These genes were selected to broadly cover the A4 subfamily tree and to contain diversity at the specificity-determining positions. Sequence identity to Cel5A_Tma of each gene is depicted with a black line on the plot, and the amino acid identities of the six specificity-determining positions are shown at right. b, the pattern of specificity changes in Cel5C_Cth and Cel5A_Eec from subfamily A4 in comparison with the corresponding mutations in Cel5A_Tma of N20A, E23A, P53A, H96A, E287A, and H95A, respectively (Fig. 2b). Cel5C_Cth and Cel5A_Eec are 29 and 25% identical to Cel5A_Tma, respectively, and closely match specificity patterns observed for Cel5A_Tma except the P72A mutation in Cel5C_Cth. Data in a and b are means from three independent experiments; error bars show S.D.

FIGURE 4.

Structural models of glucan- and mannan-based disaccharides in the −1 and −2 subsites (nomenclature of Davies et al. (44)) of the Cel5A_Tma crystal structure (PDB ID 3MMW (10)). Glucose and mannose differ in the configuration of the OH-C2 groups, which are labeled in orange. Hydrogen bonds between glucan (a) and mannan (b) substrates and Cel5A_Tma and between residues in the six-residue motif are shown with black dashed lines, and the hydrogen-acceptor distances are labeled; hydrogen bonds between OH-C2 and Cel5A_Tma are labeled in orange for clarity. The orientations of the substrates were modeled based on the orientation of cellotriose in the Cel5A_Bag crystal structure (45). Further details about the hydrogen bonding geometries are provided in supplemental Table S4.