The Investigation Of Structural And Functional Relationships Of Inulin Fructotransferase And Difructose Anhydride Hydrolase

Inulin that extensively exists in nature is a kind of fructan,which stores energy for organisms as starch.Usually,to provides energy for organism,it will be decomposed to small molecule.There are many metabolic paths for inulin,one of which is decoposed by inulin fructotransferase(IFTase)and the last product is difructose anhydride(DFA)which contains DFA-Ⅰ and DFA-Ⅲ.According to the types of products,IFTase is designated as two types including type-I IFTase(IFTase-Ⅰ)and type-Ⅲ IFTase(IFTase-Ⅲ).Simutaneously,DFA-Ⅲ can be decomposed to inulobiose by DFA-Ⅲ hydrolase.As we all know,the structure of biomacromolecule determines its function,that is,the specific structure of enzyme determines its catalytic function.In the field of enzymology,the structure,function,and catalytic mechanism is the perpetual subject.On the basis of these facets,rational design,modification,even mimetic enzyme can be implemented to obtain new enzyme with higher efficiency.To date,only the structure of IFTase-Ⅲ has been reported but not DFA-Ⅲase and IFTase-Ⅰ.Therefore,the investigation of IFTase-Ⅰ and DFA-Ⅲase structures is not only benefit for the comprehension of thier catalytic mechanism,functional and structural relationship but also benefit for the understanding of difference among these three enzymes,which provides theoretic basis for the rational design,functional modification of IFTase and DFA-Ⅲase.In this dissertation,new IFTase-Ⅰ and DFA-Ⅲase were screened and indentified.Their catalytic mechanisms were revealed by their crystal structures.The functional and structural relationships of(among)IFTase-Ⅰ,IFTase-Ⅲ,and DFA-Ⅲase were also elucidated by the structures.Simultaneously,this dissertation also reveals how inulin is metabolized in nature from molecular level.On the basis of DFA-Ⅲase’s structure,one mutant C387 A was rationally designed and it shows high catalytic level for DFA-Ⅲ.The mechanism of this improvement of catalytic activity was resolved by the analysis of the crystal structure of C387 A,which provides the theoretic basis for the further molecular modification of DFA-Ⅲase.The main contents and results are listed as follows:1.An IFTase from Clostridium clostridioforme AGR2157 was purified and designated as Cc IFTase-Ⅰ.The molecular mass assayed by SDS-PAGE and native molecular mass determined by gel filtration spectrum are 42 and 128 k Da,respectively,which indicates that Cc IFTase-Ⅰ is a homotrimer.The enzyme shows optimum activity at p H 5.5 and 50 °C,under which the enzyme activity is 2076 U mg-1.The kinetic parameter Km and kcat/Km are 0.42 mmol L-1 and 130(mmol L-1)-1 s-1,respectively.In comparison with the reported IFTase-Ⅰ,Cc IFTase-Ⅰ has higher catalytic efficiency.2.The DFA-Ⅲase gene was successfully obtained from Arthrobacter aurescens SK8.001 and submitted to Gen Bank with the accession number of KR534324.The plasmid containing this gene was constructed as p ET-22b-Aa DFA-Ⅲase and introduced to Escherichia coli(E.coli)system.The expressed and purified recombinant protein was designated as Aa DFA-Ⅲase with molecular mass of 49 k Da assayed by SDS-PAGE and natiove molecular mass of 145 k Da.The product identified as inulobiose by 13C-NMR,which indicates that the purified enzyme is DFA-Ⅲase.The optimum reaction condition of Aa DFA-Ⅲase is p H 5.5 and 55 °C,under which the enzyme activity is 232 U mg-1 and Km of 30.7 mmol L-1.Additionally,the structure,active center,and critical catalytic residue was investigated by homologous modeling,molecular docking,and site-directed mutagenesis.The result shows that the active residues of active center between Aa DFA-Ⅲase and IFTase-Ⅲ are the same except the residues from the lid of Aa DFA-Ⅲase,which indicates that the two enzymes have evolutionary relationship to some extent.3.A DFA-Ⅲase from Arthrobacter chlorophenolicus A6 was purified and designated as Ac DFA-Ⅲase.The enzyme shows optimum activity at p H 6.5 and 55 °C,under which the enzyme activity is 101.25 U mg-1.At 20 °C,the crystal of Ac DFA-Ⅲase was obtained and the optimum condition for crystallization is 0.1 mol L-1 sodium malonate(p H 4.2)and 6 % PEG 3350(W/V).The cryal data was obtained by X-ray diffraction and two structures were resolved,including Ac DFA-Ⅲase,and Ac DFA-Ⅲase in complex with DFA-Ⅲ.The structure of Ac DFA-Ⅲase is very similar to that of the reported IFTase-Ⅲ except an additional lid from Ac DFA-Ⅲase.Combining with biochemical experiment,the catalytic mechanism was resolved,which reveled that a typical inverting mechanism is used by Ac DFA-Ⅲase.Moreover,the ability to decompose inulin by Ac DFA-Ⅲase was found for the first time,which is different from the reported DFA-Ⅲases.This indicates that DFA-Ⅲase with IFTaseⅢ activity decomposes inulin to DFA-Ⅲ which is further hydrolyzed to inulobiose by DFAⅢase itself,that is,Ac DFA-Ⅲase has a function of sequentially catalyzing inulin.The structure of Ac DFA-Ⅲase in complex with GF2 was obtained,which indicates that the active center and ligand are same with those of IFTase-Ⅲ.Additionally,Ac DFA-Ⅲase loses its ability to hydrolyzing DFA-Ⅲ after deleting its lid.The structure of Ac DFA-Ⅲase without the lid is the same with that of IFTase-Ⅲ,as well as their ligands.These results indicate that the catalytic mechanisms of Ac DFA-Ⅲase and IFTase-Ⅲ to inulin are the same.4.Given a low catalytic efficiency,the rational design was implemented based on the resolved strucuture.The residue of Cys387 in the front end of lid is over ligand.After mutating it to Ala387,the relative enzyme activity of C387 A was increased to 185%.The crystal data of C387 A and its complex with DFA-Ⅲ were obtained,the resolved structure shows that the side chain of Ala387 in C387 A is shorter than that of Cys,which contributs to larger space over ligand.This space facilitates the ligand’s going up,which leades to the increase of catalytic efficiency.5.The crystal of Cc IFTase-Ⅰ was obtained and the conditions are: 0.45 mol L-1 ammonium tartrate dibasic,0.1 mol L-1 sodium acetate trihydrate at p H 4.45 and 20 °C.The resolved structure shows that the structures of Cc IFTase-Ⅰ and IFTase-Ⅲ are very similar,and the position and conformations of critical active residues are the same.However,the auxiliary residues are slightly different.This difference probably contributes the different orientation of substrate,which finally leads to the different products,that is,two isomers of DFA-Ⅰ and DFA-Ⅲ.