Structural and Functional Snapshots of a Broad-Specificity Endoglucanase from Thermogutta terrifontis for Biomass Saccharification.

Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-D-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70-80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80 °C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α)8-barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries.