| Plants or microorganism in nature occupy an important position in the survival and development of human beings,and various substances with biological activity can be synthesized in their bodies.This dissertation mainly studies the structure and function of enzymes that generate two biologically active substances,namely,fucoidan lyase(sulfated fuco-glucuronomannan,FdlA)from marine bacteria and tocopherol cyclase(VTE1)from Arabidopsis thaliana.The first part of this thesis is to study the structure and function of FdlA.Fucoidan is a polyanionic sulfated fucoidan with various biological activities from brown algae.It can be cleaved by fucoidan degrading enzymes,producing low molecular weight fucoidan that is more suitable for pharmacological use.Most of the reported fucoidan-degrading enzymes are glycoside hydrolases,which have been well studied for their structures and catalytic mechanisms.However,little is known about the catalytic mechanism of fucoidan lyase due to lack of structural information.FdlA is a novel polysaccharide lyase secreted from the marine bacterium Flavobacterium sp.SA-0082.Different from the widely reported fucoidan hydrolase,it cleaves fucoidan from Kjellmaniella crassifolia in a lytic manner,producing three types of trisaccharide molecule with unsaturated double bonds.As the full-length FdlA tends to aggregate when expressed in E.coli,we constructed a truncated version of FdlA only consisting of the N-terminal catalytic domain(residue 25～471)after sequence analysis,namely FdlA-NTD.The biochemical results showed that FdlA-NTD possesses the same enzymatic properties as its full-length native protein,and nine crystal structures of FdlA-NTD(including wild-type and mutant)were determined,and enzymatic assays and affinity experiments were carried out.The structure shows that FdlA-NTD adopts a right-handed parallel β-helix fold with an unusual cysteine ladder,moreover,a long groove and a unique basic pocket constitute substrate-binding site of FdlA-NTD.Since the structure of the complex bound to the substrate was not obtained,we obtained models of FdlA-NTD bound to different oligosaccharides by molecular docking,and analyzed the inactive mutant structures.Based on the above results,a possible catalytic mechanism of FdlA-NTD was proposed,that is,positively charged residues neutralize the C-5 carboxyl group at the+1 subsite,while Lys141 and Tyr242 function as catalytic base and catalytic acid,respectively.In addition,FdlA-NTD utilizes catalytic residues different from other β-helix polysaccharide lyases,and the sequence homology between them is low,indicating that FdlA-NTD is likely to represent a novel polysaccharide lyases family.This study provides a molecular basis for an in-depth understanding of the efficient degradation of fucoidan by fucoidan lyase.The second part of this thesis is to study the structure and function of VTE1.VTE1 is a key enzyme in the synthesis of vitamin E in plants and catalyzes the formation of aromatic rings from different phytohydroquinone intermediates,finally synthesizes the corresponding tocopherol.We found that VTE1 exists as a monomer at high imidazole concentration,while the absence of imidazole will cause VTE1 to form a uniform oligomeric state.Finally,VTE1 dodecamer with an overall structure of 3.9 ? was obtained by cryo-electron microscopy.The structure shows that each VTE1 protein consists of two β-barrels,and y-tocopherol is present in the deep cavity between two β-barrels.The possible catalytic reaction information was obtained by analyzing the structure of the complex and the results of docking.This study provides theoretical support for increasing the content of vitamin E in plants,and deepens the understanding of the structure and function of cyclase. |
