β-Mannoside hydrolases from clan A – Structural enzymology, subsite engineering and transglycosylation

Detta är en avhandling från Department of Chemistry, Lund University

Sammanfattning: β-Mannoside hydrolases are enzymes that are involved in conversion of the major soft-wood hemicellulose O-acetyl-galactoglucomannan, an important renewable biomass resource. They belong to clan A of glycoside hydrolases and share a common (β/α)8-TIM barrel fold. For their catalysis, they use a two-step retaining double displacement mechanism, and may in addition to hydrolysis also perform transglycosylation, i.e. synthesis of new glycosidic linkages. Although sharing the same fold, catalytic mechanism and basic enzymatic activity there are differences in fine-tuned function, which often derives from differences in the active site subsite organisation. The aim of this thesis was to reveal knowledge about molecular structure and functional properties of β-mannosidases from GH2 and β-mannanases from GH5 and GH26, with focus on substrate binding and transglycosylation. This is of value for further studies but also when selecting enzymes with specific functions for certain applications. This thesis contributes with three crystal structures of GH5 and GH26 β-mannanases. For analysis of the binding of oligosaccharides in the active site, a new method was developed. Quantitative product analysis using HPAEC-PAD is combined with MALDI-TOF MS analysis of reactions performed in 18O-labelled water, which enables mapping of active site subsites involved in substrate binding. A combined phylogenetic and biochemical analysis of GH2 β-mannosidases fom Aspergilli showed that they cluster in two clades (A and B), where clade A and B β-mannosidases appeared to have different functions. The studied microbial GH26 β-mannanases are from different environments (soil, dung and human gut). They show differences in modularity, function, structure and subsite organisation. None of the GH26 β-mannanases displayed transglycosylation activity. Rational engineering, shifting affinity from subsite -3 to subsite -2, however introduced weak transglycosylation with saccharide acceptors for CfMan26A from Cellulomonas fimi. The studied GH5 β-mannanases are of fungal origin. They all showed transglycosylation activity. A subsite +2 mutant (R171K) of TrMan5A from Trichoderma reesei could not transfer to saccharides but the ability of transfer to alcohol acceptors was retained. AnMan5B from Aspergillus nidulans had an additional Trp in the +2 subsite and showed comparably high transglycosylation with saccharides, but could not transfer to alcohols. Thus, strong binding in the aglycone region appeared to be an important factor for transglycosylation with saccharides but not necessarily with other acceptors, such as alcohols.

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