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.
Highlights
Sustainable, biological solutions to the growing climate and energy crises have been the subject of increasing interest in the last 2 decades
The ;5-Å distance between the side chains of these residues is consistent with the retaining mechanism of glycoside hydrolases in GH5 [28]
It should be noted that the full polypeptide sequences of some of these proteins include carbohydrate-binding modules, dockerin domains, and additional enzyme domains (Fig. 1C), but the experimental design for this study included only the GH5_4 enzyme domains
Summary
Sustainable, biological solutions to the growing climate and energy crises have been the subject of increasing interest in the last 2 decades. Broad-specificity glycoside hydrolases (GHs) may replace many specialized enzymes with fewer, more flexible catalysts, increasing sugar yield and reducing enzyme variability between feedstocks [2]. One such family rich in broad-specificity cellulases is GH5. Variability of function is thought to occur through differences in the loop regions connecting the b-strands to the a-helices, at the C-terminal side of the barrel where the active site is built [8, 9]. Many efforts have been directed toward understanding how the sequence and structure of a particular GH5_4 enzyme dictate its substrate selectivity [9, 10, 12, 13, 16,17,18,19]
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