Structure and Function of Beta-Mannanases from Glycoside Hydrolase Clan A. A Study of Hemicellulases from Microbes and a Mollusc

Sammanfattning: Hemicellulose is the second most abundant group of renewable polysaccharides. Mannans can be found as the major hemicellulose in softwood and as storage polysaccharides in a range of plants. In this work, structural and functional characterisations of family 5 and 26 endo-1,4-b-mannanases (b-mannanases) have been performed. These families belong to the large and diverse glycoside hydrolase clan A, where the members exhibit the (b/a)8-barrel fold and a double displacement mechanism. b-mannanases hydrolyse the internal mannosidic bonds of mannan based polysaccharides. With increased knowledge in mannan degradation enzymology, we will be able to envision novel modifications of mannan-based polysaccharides for use in various applications. Furthermore, the transglycosylation capacity of these enzymes may be used for the synthesis of novel glyco-conjugates. This work has focused on three b-mannanases from different organisms exemplifying the diversity of this type of enzymes: CfMan26A from the bacterium Cellulomonas fimi (family 26), MeMan5A from the mollusc Mytilus edulis (family 5, subfamily 10) and HjMan5A from the filamentous fungus Hypocrea jecorina (family 5, subfamily 7). The three enzymes also show diversity in their respective modular organisation, CfMan26A consists of five modules, HjMan5A of two modules and MeMan5A consists of a sole catalytic module. Furthermore, CfMan26A has a mannanbinding module while HjMan5A has a cellulose-binding module. These modules are important in the overall function of the enzymes. The 3D-structures of CfMan26A and MeMan5A catalytic modules were solved by X-ray crystallography. A previously unknown immunoglobulin module was positioned in CfMan26A between the catalytic and the mannan-binding modules. MeMan5A is the first b-mannanase from an animal to be structurally characterised. Differences to the other family 5 b-mannanases were observed and we proposed that this b-mannanase should be assigned into a new subfamily (subfamily 10). Product patterns from mannotetraose hydrolysis experiments suggested that the three characterised enzymes had different subsite topologies. Structural and kinetic data was used to conduct partial subsite mapping. The data indicated that the distal +2 subsite has less importance in CfMan26A compared to MeMan5A and HjMan5A. Furthermore, CfMan26A and HjMan5A have five important subsites while MeMan5A uses at least six important subsites to achieve efficient hydrolysis. Interestingly, CfMan26A showed no indications of transglycosylation. Decreased level of transglycosylation was also observed for a +2 subsite mutant of HjMan5A, indicating the importance of the +2 subsite in transglycosylation. The increased knowledge in glycoside hydrolase specificity could assist us in designing novel enzymes that can recognise defined motifs of complex polysaccharides with high accuracy and efficiency.