Unique features of the bifunctional GH30 from Thermothelomyces thermophila revealed by structural and mutational studies.

Fungal xylanases belonging to family GH30_7, initially categorized as endo-glucuronoxylanases, are now known to differ both in terms of substrate specificity, as well as mode of action. Recently, TtXyn30A, a GH30_7 xylanase from Thermothelomyces thermophila, was shown to possess dual activity, acting on the xylan backbone in both an endo- and an exo- manner. Here, in an effort to identify the structural characteristics that append these functional properties to the enzyme, we present the biochemical characterization of various TtXyn30A mutants as well as its crystal structure, alone, and in complex with the reaction product. An auxiliary catalytic amino acid has been identified, while it is also shown that glucuronic acid recognition is not mediated by a conserved arginine residue, as shown by previously determined GH30 structures.

Structural and enzymatic characterization of a glycoside hydrolase family 31 α-xylosidase from Cellvibrio japonicus involved in xyloglucan saccharification.

The desire for improved methods of biomass conversion into fuels and feedstocks has re-awakened interest in the enzymology of plant cell wall degradation. The complex polysaccharide xyloglucan is abundant in plant matter, where it may account for up to 20% of the total primary cell wall carbohydrates. Despite this, few studies have focused on xyloglucan saccharification, which requires a consortium of enzymes including endo-xyloglucanases, α-xylosidases, ß-galactosidases and α-L-fucosidases, among others. In the present paper, we show the characterization of Xyl31A, a key α-xylosidase in xyloglucan utilization by the model Gram-negative soil saprophyte Cellvibrio japonicus. CjXyl31A exhibits high regiospecificity for the hydrolysis of XGOs (xylogluco-oligosaccharides), with a particular preference for longer substrates. Crystallographic structures of both the apo enzyme and the trapped covalent 5-fluoro-ß-xylosyl-enzyme intermediate, together with docking studies with the XXXG heptasaccharide, revealed, for the first time in GH31 (glycoside hydrolase family 31), the importance of a PA14 domain insert in the recognition of longer oligosaccharides by extension of the active-site pocket. The observation that CjXyl31A was localized to the outer membrane provided support for a biological model of xyloglucan utilization by C. japonicus, in which XGOs generated by the action of a secreted endo-xyloglucanase are ultimately degraded in close proximity to the cell surface. Moreover, the present study diversifies the toolbox of glycosidases for the specific modification and saccharification of cell wall polymers for biotechnological applications.

Structure-function relationships of a catalytically efficient beta-D-xylosidase.

Beta-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. 1H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl- beta-D-xylopyranoside (4NPX) and 1,4-beta-D-xylobiose (X2). Progress curves of 4-nitrophenyl-beta-D-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of kcat and kcat/Km decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites -1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond -1 are blocked by protein and subsites beyond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode kcat and kcat/Km by <103 and to a similar extent for substrates 4NPX and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs kcat/Km with pKa values of 5.0 and 7.0 assigned to D14 and E186, respectively. kcat(4NPX) has a pKa value of 7.0 and kcat(4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, "dianionic" enzyme form (D14-E187-) binds 4NPX but not 4NPA.

Ultrastructural diversity of the cellulase complexes of Clostridium papyrosolvens C7.

Transmission electron microscopy was used to investigate the ultrastructural features of diverse cellulase and cellulase-xylanase multiprotein complexes that are components of the cellulase-xylanase system of Clostridium papyrosolvens C7. The multiprotein complexes were separated by anion-exchange chromatography into seven biochemically distinguishable fractions (F1 to F7). Most individual F fractions contained, in relatively large numbers, an ultrastructurally recognizable type of particle that occurred only in smaller numbers, or not at all, in the other F fractions. It is suggested that these ultrastructurally distinct particles represent the biochemically distinct multiprotein complexes that constitute the cellulase-xylanase system of C. papyrosolvens C7. Some of the particles consisted of tightly packed globular components that appeared to be arranged in the shape of a ring with conical structures pointing out along its axis. Other particles had triangular, polyhedral, or star shapes. The major protein fraction (F4) almost exclusively contained particles consisting of loosely aggregated components, many of which appeared to be arranged along filamentous structures. The ultrastructural observations reported here support our previous conclusion that the cellulase-xylanase system of C. papyrosolvens C7 comprises at least seven different high-molecular-weight multiprotein complexes. Furthermore, results of this and earlier studies indicate that the interactions between C. papyrosolvens C7 and cellulose are different from those that have been described for Clostridium thermocellum.

A common protein fold and similar active site in two distinct families of beta-glycanases.

The structure of Clostridium thermocellum endoglucanase CelC, a member of the largest cellulase family (family A), has been determined at 2.15 A resolution. The protein folds into an (alpha/beta)8 barrel, with a deep active-site cleft generated by the insertion of a helical subdomain. The structure of the catalytic core of xylanase XynZ, which belongs to xylanase family F, has been determined at 1.4 A resolution. In spite of significant differences in substrate specificity and structure (including the absence of the helical subdomain), the general polypeptide folding pattern, architecture of the active site and catalytic mechanism of XynZ and CelC are similar, suggesting a common evolutionary origin.

Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei.

Three-dimensional structures of two major endo-1,4-xylanases, XYNI and XYNII from Trichoderma reesei, have been determined by X-ray crystallography. The amino acid sequences of both enzymes are highly homologous (identity approximately 50%), and both XYNI and XYNII exist as a single domain that contains two mostly antiparallel beta-sheets which are packed against each other. The beta-sheet structure is twisted, forming a cleft where the active site is situated. Two glutamic acids in the cleft, Glu75 and Glu164 in XYNI as well as Glu86 and Glu177 in XYNII, are most likely involved in catalysis. Inspection of the structures reveals that the width of the active site cleft and the number of subsites are different in XYNI and XYNII. The active site is narrower in XYNI and probably contains only three subsites, whereas the number of subsites in XYNII is most likely five. Variations in the surroundings of catalytic residue Glu164XYNI/Glu177XYNII are thought to explain the pH optimum differences observed in XYNI and XYNII.