Structural Analyses And Structure-Function Studies Of The Key Enzymes To Produce D-Allulose And D-Allose Using D-Fructose

Rare sugars are the monosaccharides and their derivatives that rarely exist in nature.They are widely used in food and pharmaceutical industries due to the unique biological and physiological functions.D-Allulose and D-allose are two kinds of rare monosaccharides among them,which have 70%and 80%of the sweetness of sucrose,ultra-low the energy,and no side effects,respectively.Thus,both have great potential uses in the sweetener and lean healthcare.However,rare sugars are difficult to extract from natural source and chemically synthesize.Therefore,the bioconversion of rare sugars has attached more attention.D-Allulose and D-allose can be enzymatically produced by ketose 3-epimerase and L-rhamnose isomerase using the cheap and abound D-fructose according to the Izumoring strategy,respectively.Both are the key enzymes to produce D-allulose and D-allose.In this study,five kinds of ketose 3-epimerases were mined and identified that they can covert D-fructose to D-allulose,the apo-form and co-complex structures with different substrates of two DAEs have been determined.The molecular modifications were carried out based on the crystal structures.At the same time,two L-rhamnose isomerases were also characterized that they can covert D-allulose to D-allose,the apo-form of both have been determined and the mechanism of thermostability have been revealed,respectively.The main conclusions are summarized as follows.（1）Five kinds of new genes encoded ketose 3-epimerases were screened successfully,which can covert D-fructose to D-allulose.They are four kinds of D-allulose 3-epimerases from Sinorhizobium fredii CCBAU 83666,Staphylococcus aureus,Agrobacterium sp.SUL3,and Halanaerobium congolense,respectively（DAE:Sf DAE,Sa DAE,a DAE,and Hc DAE）,and one D-tagatose 3-epimerase from Sinorhizobium sp.RAC02（DTE;s DTE）.They were expressed successfully in the soluble form in E.coli BL21（DE3）.Highly purified proteins were obtained.The enzymatic properties of five recombinant ketose 3-epimerases were characterized,the optimum p H and temperatures of them varied from 7.5 to 8.0,and 50 to70°C,respectively.Sf DAE showed highest thermostability among them.All of them were metal-dependent,and the optimum ions were Mg²⁺for Sf DAE,Sa DAE,Hc DAE,and a DAE,Mn²⁺for s DTE,respectively.（2）The data of apo-form and co-complex structures with different substrates of Sf DAE and a DAE were collected using X-ray crystallography.The structures with high resolutions were obtained after further analysis and refinement,the resolutions of apo-form and co-complex structures of Sf DAE with D-fructose,D-allulose,D-tagatose,and D-sorbose were1.55（?）,1.70（?）,1.80（?）,2.10（?）,and 1.88（?）,respectively.The resolutions of apo-form and co-complex structures of a DAE with D-fructose and D-allulose were 2.05（?）,2.12（?）,and 2.32（?）,respectively.Aα-helix,which was the lid（α’8）of Sf DAE with open/closed state was observed in the crystal structure.The lid was in the closed state after binding substrate,and the residue M15 showed a significant change that it was away from active pocket.This lid was important for the catalytic activities of DAE family by substitute,deletion,and mutation of the residues of the lid.The mechanism that DAE family preferred D-allulose as the optimum substrate was revealed,which was due to the more interactions between DAEs with D-allulose than D-fructose by forming more hydrogen bonds.（3）The mutants of Sf DAE with higher thermostability were obtained based on the crystal structures,the mutants of M15K,A254C,and M15K/A254C exhibited an increase of the Tm value by 3.5,2.5,and 5.5°C,respectively.The models of structures of Sa DAE and Hc DAE were analyzed,and molecular modifications were carried out,respectively.Some mutants with improved properties were obtained.The redesigned mutant V105A from Sa DAE showed an improvement of relative activity toward D-fructose of 68%based on sequence alignment by site-directed mutation.The triple mutant S191D/M193E/S213C of Sa DAE with higher thermostability was also obtained.It showed an increase of the Tm value by 7.2°C than that of wild type.Saturation mutagenesis of the putative residues of Hc DAE responsible for substrate recognition was performed,and it yielded the triple mutant Y7H/C66L/I108A with improved activity toward D-fructose（3.45-fold of wild-type enzyme）.The mutant of K260C/R156C with introduced disulfide bridges between intersubunits exhibited an increase of Tm value by 3.5°C.The combined mutant Y7H/C66L/I108A/R156C/K260C exhibited a half-half（t1/2）of 5.2 h at 70°C and an increase of the Tm value by 6.5°C than that of wild type（6.3 h and 79.5°C）.（4）The enzymatic properties of L-rhamnose isomerases from the thermophilic organism Dictyoglomus turgidum DSM 6724 and Thermotoga maritima MSB8（LRI:Dt LRI and Tm LRI）were characterized,the optimum conditions for Dt LRI and Tm LRI were p H 8.0 and70°C,p H 8.0 and 80°C,respectively.The Tm values of Dt LRI and Tm LRI were 57.5 and75.5°C,respectively.Thus,Tm LRI showed a higher thermostability.（5）The crystal structure of apo-form of Dt LRI and Tm LRI were obtained using X-ray crystallography.The resolutions of apo-form Dt LRI and Tm LRI were 2.90（?）and 2.74（?）,respectively.The mechanisms of thermal stability of Tm LRI were revealed based on the crystal structure.Tm LRI has higher contents of amino acids of alanine,proline,arginine,and glutamate than that from Dt LRI and LRI from Bacillus halodurans（Bh LRI）.Those amino acids were reported to contribute to the thermostability of enzymes.In general,the N-terminus and C-terminus of thermophilic enzymes have interactions with around residues.Therefore,it cannot be degraded due to the stable conformation under high temperature.Theα-helix（α0）at N-terminus of Dt LRI and Bh LRI was present with a higher B-factor,while that was absent in Tm LRI.The Tm values of Dt LRIΔN after deletion ofα0 at N-terminus exhibited an increase of the Tm value（62.5°C）by 5°C than that of wild-type Dt LRI.