A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis

Abstract

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

Keywords: GH5; bioenergy; cellulase; endoglucanase; glycoside hydrolase; polysaccharide; substrate specificity; xyloglucanase.

Figure 1.

Structural, biological, and phylogenetic overview of GH5_4 enzymes. A, tertiary structure and the location of polysaccharide binding modeled from glucan chain of XG fragments in the 2JEQ (17) and 4W88 (16) structures. Aromatic residues in the binding cleft are displayed as spheres. Loops are labeled and color-coded. B, close-up of the binding cleft showing surface interactions with substrate chain: Trp (green), Tyr (orange), and catalytic Glu residues (red). The numbering of the sugar-binding subsites is indicated. C, source organism, accession code, and native domain structure of the 10 enzymes in this study. Domain annotations were obtained from NCBI, UniProt, and CAZy. Blue, GH5_4 domain; green, CBM; yellow, carbohydrate esterase; orange, dockerin; purple, BACON domain. D, phylogenetic tree of clades and subclades of GH5_4 (yellow, red, and green wedges) and closely related subfamilies (numbered gray wedges).

Figure 2.

Cross-sections of GH5_4 enzymes showing the active-site cleft at the approximate position of the active site. The view is from the positive sugar-binding subsites toward the negative subsites. The variety of cleft shapes owes mainly to length differences in loops 4 (left) and 6 (right). The locations of the conserved catalytic Glu nucleophile (red) and Glu proton donor (blue) are shown for 4IM4, and the locations of aromatic residues (Trp in green and Tyr in orange) are highlighted in each structure.

Figure 3.

Aromatic contacts for XG binding modeled by alignment with PDB 4W88 (16) and 2JEQ (17). Left panels, negative subsites; right panels, positive subsites. A and B, 4IM4; C and D, 6XSO; E and F, 6XRK; G and H, 6XSU. The sugar colors are as follows: glucose (yellow), xylose (cyan), and galactose (magenta). The aromatic residue colors are as follows: Trp (green), Tyr (orange), and Phe (blue).

Figure 4.

Interactions of loop 3 in 6XSO predicted from the XG ligands provided by alignment with 2JEQ. Potential polar contacts between side chains of Lys-129 and Asp-130 and to galactose branch in XG are shown. A network of possible intersugar hydrogen bonds is also indicated. The sugar colors are as follows: glucose (yellow), xylose (cyan), and galactose (magenta). Distances are reported in Å.

Figure 5.

Catalytic efficiency shown as a plot of turnover numbers (k_cat) and Michaelis constants (K_m) for reaction of GH5_4 with GM (yellow triangles), XG (purple circles), lichenan (L, blue squares), and xylan (X, red circles). The slope of the imaginary line drawn from the origin out to each point yields the catalytic efficiency. Error bars, one S.D. of three simultaneous experimental replicates. Dashed lines, linear correlation functions for each substrate, with R² indicated.

Figure 6.

Turnover numbers of GH5_4 measured at 10 mg/ml substrate. Substrates were as follows: mannan (M), phosphoric acid–swollen cellulose (P), beechwood xylan (X); tamarind XG, lichenan (L), and konjac GM. Error bars, one S.D. over three simultaneous experimental replicates.

Figure 7.

End products of oligo- and polysaccharide hydrolysis by 6UI3 as determined by quantitative NIMS. A, cellulose-based substrates. B, mannose-based substrates. Error bars, one S.D. of three simultaneous experimental replicates.

Figure 8.

Model of cellulose and mannan cleavage specificity in the 6UI3 active-site cleft. A, structure of 6UI3 with β-(1,4)-glucan chain of XG modeled in to approximate the binding of cellohexaose. C-2 carbon atoms are shown as magenta spheres to highlight the location of stereochemical difference between glucose and mannose. Clamp-forming loop 4 and loop 6 are highlighted in blue. Green, Trp; orange, Tyr. B, schematic of 6UI3-binding cleft, highlighting sugar subsites and key binding (Trp-42, Trp-159, Tyr-227) and catalytic (Glu-152, Glu-271) residues. Trp-42 is hypothesized to play an important role in the registry shift from cellulose to mannan binding at the −3 subsite.