Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 ß-Xylosidases.

The glycoside hydrolase family 39 (GH39) is a functionally expanding family with limited understanding about the molecular basis for substrate specificity and extremophilicity. In this work, we demonstrate the key role of the positive-subsite region in modulating substrate affinity and how the lack of a C-terminal extension impacts on oligomerization and structural stability of some GH39 members. The crystallographic and SAXS structures of a new GH39 member from the phytopathogen Xanthomonas citri support the importance of an extended C-terminal to promote oligomerization as a molecular strategy to enhance thermal stability. Comparative structural analysis along with site-directed mutagenesis showed that two residues located at the positive-subsite region, Lys166 and Asp167, are critical to substrate affinity and catalytic performance, by inducing local changes in the active site for substrate binding. These findings expand the molecular understanding of the mechanisms involved in substrate recognition and structural stability of the GH39 family, which might be instrumental for biological insights, rational enzyme engineering and utilization in biorefineries.

Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43.

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-ß-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-ß-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related ß-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the ß-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.

The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans.

BACKGROUND: Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). RESULTS: In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the - 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. CONCLUSIONS: In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization.