Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

Abstract

Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

Molecular Mechanisms Associated with Xylan Degradation by Xanthomonas Plant Pathogens

“…12) showing conserved Ϫ3, Ϫ2, and Ϫ1 subsites (Fig. 6), as reported in previous crystallographic studies with GH10 xylanases (40,47,48,53). It is worthy of mention that the xylosyl moiety occupying the Ϫ3 subsite is not contacting any protein residue with its orientation determined by steric impositions of the glycosidic bond within the trisaccharide (Fig.…”

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

“…Currently, xylanase has been attracting much attention due to its wide biotechnological applications in the food, animal feed, pulp and paper, and textile industries and in the deconstruction of lignocellulose for biofuel production (2)(3)(4)(5)(6). On the basis of the catalytic domains, xylanases are mainly classified into glycoside hydrolase (GH) families 5,8,10,11,30, and 43 (7); GH10 and GH11 xylanases are the best studied.…”

“…To obtain thermostable xylanases, one strategy is to search for novel xylanases from extremophiles. A number of thermostable GH10 xylanases have been reported for thermophilic Thermotoga maritima MSB8 (90°C) (10), Thermotoga petrophila RKU-1 (90°C) (11), and Thermobifida alba UL JB1 (80°C) (12) and for acidomesophilic Bispora sp. strain MEY-1 (85°C) (13).…”

“…strain MEY-1 (85°C) (13). The available sequences and structures of these thermophilic xylanases (i.e., 1VBU and 3NIY [10,11]) provide valuable information for better understanding of the structural and functional characters of thermostable enzymes.…”

Identifying critical unrecognized sugar–protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

“…X-ray crystallography of X2-xylanase crystals shows binding across subsites À2 and À1, corroborated by ITC studies [14,19,42,43,45,46]. The À2/À1 subsites together have been dubbed the substrate recognition area, as opposed to the +1/+2 subsites, which are referred to as the product release area [43].…”