Two new major subunits in the cellulosome of Clostridium thermocellum: xyloglucanase Xgh74A and endoxylanase Xyn10D

Abstract

The structure and enzymic activity of xyloglucanase Xgh74A and endoxylanase Xyn10D, components in the cellulosomes of cellulose-grown Clostridium thermocellum, were determined. Xyn10D is a thermostable endo-1,4-β-xylanase with a module composition identical to Xyn10C (CBM22-GH10-Doc). It hydrolyses xylan and mixed-linkage 1,3-1,4-β-glucan with a temperature optimum of 80 °C. Xyloglucanase Xgh74A contains a catalytic module of GHF74 in addition to a C-terminal dockerin module. It hydrolyses every fourth β-1,4-glucan bond in the xyloglucan backbone, thus producing decorated cellotetraose units. Its low activity on CMC and lack of activity on amorphous cellulose indicates recognition of the xylosidic side chains present in xyloglucan, which is readily hydrolysed (295 U mg−1). The pattern of the hydrolysis products from tamarind xyloglucan resembles that of other GHF74 xyloglucan endoglucanases. The data indicate that Xgh74A and Xyn10D contribute to the in vivo degradation of the hemicelluloses xyloglucan and xylan by the cellulosome of C. thermocellum. Xgh74A is the first xyloglucanase identified in C. thermocellum and the only enzyme in the cellulosome that hydrolyses tamarind xyloglucan.

Characterization of All Family-9 Glycoside Hydrolases Synthesized by the Cellulosome-producing Bacterium Clostridium cellulolyticum

“…Cel9X had no detectable activity on the hemicellulosic polysaccharides barley glucan, xylan, arabinan, and laminarin, thus suggesting this enzyme is highly specific of xyloglucan. This activity pattern is very unusual for a GH9 enzyme and also differs from typical xyloglucanases belonging to family-44 like CelB from R. flavefaciens FD1 (32) or family 74 like Xgh74 from C. thermocellum (33), which are described to exhibit some activity on barley glucan and/or CMC. Interestingly, Cel9X resembles (53% sequence identity) the N terminus part of CelJ from C. thermocellum made of CBM30-Ig-GH9 (also called Cel9D), which was shown to be an endocellulase (5) having significant activities on barley glucan, lichenan, and xyloglucan (7).…”

Crystal Structures of Clostridium thermocellum Xyloglucanase, XGH74A, Reveal the Structural Basis for Xyloglucan Recognition and Degradation

“…The recent enzymatic characterization of the endoxyloglucanase, Xgh74A, shows that the enzyme hydrolyzes the glycosidic bond of the unbranched glucosyl residues in xyloglucan, to yield XXXG, XLXG (or XXLG), and XLLG oligosaccharides (19). Here we describe the crystal structure of Xgh74A both as an uncomplexed apoenzyme at 2.1 Å resolution, and that of an inactive variant, Xgh74A-D70A, with a single molecule of XLLG and one of XXLG bound in the active site cleft.…”

“…About half of the other proteins in the cellulosome are described as hemicellulolytic enzymes, highlighting the importance of these accessory enzymes in the processing of cellulosic composites. Recently, two major enzymes implicated in hemicellulose degradation by the Ct cellulosome have been characterized; an endo-␤-xylanase and a xyloglucanase (19). Xyloglucanase Xgh74A, Fig.…”

Characterization of an Endo-Processive-Type Xyloglucanase Having a β-1,4-Glucan-Binding Module and an Endo-Type Xyloglucanase from Streptomyces avermitilis

“…Both SaGH74A and SaGH74B also showed activity against xyloglucans and were therefore classified as xyloglucanases. SaGH74A and SaGH74B were slow xyloglucanases compared to other bacterial xyloglucanases (specific activity for that of Jonesia sp., 30 U/mg; for that of Geotrichum sp., 68 U/mg; for that of Clostridium thermocellum, 295 U/mg; and for that of Thermobifida fusca YX, 875 U/mg) (21,25,34,36). The enzymes released a mixture of oligosaccharides from TXG, indicating that both enzymes are endo-enzymes.…”