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.

N-glycan Utilization by Bifidobacterium Gut Symbionts Involves a Specialist ß-Mannosidase.

Bifidobacteria represent one of the first colonizers of human gut microbiota, providing to this ecosystem better health and nutrition. To maintain a mutualistic relationship, they have enzymes to degrade and use complex carbohydrates non-digestible by their hosts. To succeed in the densely populated gut environment, they evolved molecular strategies that remain poorly understood. Herein, we report a novel mechanism found in probiotic Bifidobacteria for the depolymerization of the ubiquitous 2-acetamido-2-deoxy-4-O-(ß-d-mannopyranosyl)-d-glucopyranose (Man-ß-1,4-GlcNAc), a disaccharide that composes the universal core of eukaryotic N-glycans. In contrast to Bacteroidetes, these Bifidobacteria have a specialist and strain-specific ß-mannosidase that contains three distinctive structural elements conferring high selectivity for Man-ß-1,4-GlcNAc: a lid that undergoes conformational changes upon substrate binding, a tryptophan residue swapped between the two dimeric subunits to accommodate the GlcNAc moiety, and a Rossmann fold subdomain strategically located near to the active site pocket. These key structural elements for Man-ß-1,4-GlcNAc specificity are highly conserved in Bifidobacterium species adapted to the gut of a wide range of social animals, including bee, pig, rabbit, and human. Together, our findings uncover an unprecedented molecular strategy employed by Bifidobacteria to selectively uptake carbohydrates from N-glycans in social hosts.

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.

Structural basis of exo-ß-mannanase activity in the GH2 family.

The classical microbial strategy for depolymerization of ß-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-ß-mannanases and ß-mannosidases. In this work, we describe the first exo-ß-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and ß-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 ß-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 ß-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 ß-mannosidases, Gly439 and Gly556, which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-ß-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-ß-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains.

The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.