| Xylans,including β-1,4-xylan,β-1,3-xylan and β-1,3/1,4-xylan,constitute an important organic carbon in the nature.Among xylans,β-1,3/1,4-xylan is peculiar to and widely present in marine red algae.Xylanases play important roles in xylan degradation.β-1,4-xylanases(EC 3.2.1.8)and β-1,3-xylanases(EC 3.2.1.32)have been reported to be involved in the degradation of β-1,4-xylan and β-1,3-xylan,respectively.However,no enzymes have been yet identified to specifically hydrolyze β-1,3/1,4-xylan.Discovering β-1,3/1,4-xylanases from marine bacteria is helpful for revealing the degradation and recycling of β-1,3/1,4-xylan in the ocean.It is also of great significance for the exploitation and utilization of marine red algae.In our previous study,a potential β-1,3/1,4-xylanase has been identified from a marine bacterium.In this thesis,by phylogenetic analysis and enzymatic substrate specificity and action mode analyses,a novel type of xylanases,β-1,3/1,4-xylanases were characterized,which constitutes a novel β-1,3/1,4-xylanase subfamily of glycoside hydrolase family 26(GH26).We further solved the structures of a β-1,3/1,4-xylanase and its complex with substrate.By structural and mutational analyses,the mechanism for the substrate recognition and catalysis of β-1,3/1,4-xylanases was elucidated.(1)Identification of β-1,3/1,4-xylanases secreted by marine bacteriaXyn26A is a potential β-1,3/1,4-xylanase found in our previous study.To analyze the evolutionary status of Xyn26A,a phylogenetic tree was constructed based on the amino acid sequences of Xyn26A and its homologs and representative sequences from the GH26 family.The result suggested that Xyn26A and its homologs may represent a novel β-1,3/1,4-xylanase subfamily of GH26.Five representative enzymes(Xyn26A,AlXyn26A,CeXyn26A,PpXyn26A and CaXyn26A)from this subfamily were heterologous expressed and purified.Substrate specificity analysis indicated that they could only degrade β-1,3/1,4-xylan,but were inactive for β-1,3-xylan or β-1,4-xylan,which is different from the reported β-1,3-xylanases or β-1,4-xylanases.The action mode analysis showed that these β-1,3/1,4-xylanases specifically attacked β-1,3/1,4-xylan at the β-1,4-linkages following a β-1,3-linkage towards the reducing end,revealing the specific action site of β-1,3/1,4-xylanases on β-1,3/1,4-xylan.In conclusion,we identified a novel type of xylanases,β-1,3/1,4-xylanases,which constitute a novel subfamily of GH26.(2)Structural insights into the substrate recognition and catalysis mechanism of β1,3/1,4-xylanase AlXyn26ATo reveal the structural basis for the substrate specificity of β-1,3/1,4-xylanases,five β1,3/1,4-xylanases were crystallized.The crystal structure of AlXyn26A was solved.So,the biochemical properties,structure and the mechanism for substrate recognition and catalysis ofβ-1,3/1,4-xylanase AlXyn26A were investigated.The results showed that the optimum temperature and pH of AlXyn26A were 40℃ and 7.0,respectively.AlXyn26A folds as an(β/α)8 triosephosphate isomerase(TIM)barrel,a typical fold for GH26 enzymes.AlXyn26A was most closely related to,but different in substrate specificity from β-1,3-xylanase Xyl4.Comparative structural analysis between AlXyn26A and Xy14 revealed a unique tunnelshaped catalytic cavity of AlXyn26A.The cavity is covered by two flexible lid loops with a narrow circular exit,which is noticeably different from the open cleft of Xyl4.The structure of enzyme-substrate complex,E243A-X1,3X 1,4X,was further solved,Structural and mutational analysis revealed the key amino acid residues involved in the substrate binding and catalysis of AlXyn26A.AlXyn26A interacts with substrate mainly by forming hydrogen bonds and hydrophobic stacking interactions.AlXyn26A catalyzes β-1,3/1,4-xylan in a double-displacement mechanism with residues Glu147 and Glu243 as the catalytic acid/base and the nucleophile,respectively.Two lid loops and the tunnel exit of AlXyn26A were involved in substrate binding.Therefore,we predicted that the unique tunnel-shaped cavity results in the substrate specificity of β-1,3/1,4-xylanases by specifically binding β-1,3/1,4-xylan.Multiple sequence alignment analysis and structural prediction revealed that all β-1,3/1,4xylanases possessed a similar tunnel-shaped cavity.Isothermal titration calorimetry(ITC)analysis showed that both AlXyn26A and Xyn26A could effectively bind X1,3X1,4X but neither has obvious binding capacity to β-1,3-xylotriose or β-1,4-xylotriose,which verified our prediction.In this thesis,a novel β-1,3/1,4-xylanase subfamily of GH26 was characterized.The substrate specificity,action mode and the mechanism for substrate recognition and catalysis of this novel type of xylanases were elucidated.The results are helpful for revealing the degradation and recycling of β-1,3/1,4-xylan in the ocean,laying a theoretical foundation for the exploitation of marine red algal resources. |
