Molecular Mechanisms Of Thermal Activation And Substrate Specificity In The Glycogen Debranching Enzyme From Saccharolobus Solfataricus STB09

Starch debranching enzyme（SDBE）is an important enzyme employed in starch processing.It specifically hydrolyzes theα-1,6 glycosidic bonds in starch and related polysaccharides,thereby removing branched chains in the substrate.In the production of starch sugars,SDBE can synergistically work with amylases to efficiently convert raw starch materials into starch sugars.This process not only improves product quality and yield but also leads to higher economic benefits.In the enzymatic modification of starch,SDBE can also convert starch into linear dextrin,which is used for the production of resistant starch and other products with special functions.The key to improving the conversion rate of starch raw materials lies in the use of a highly efficient debranching enzyme.Such an enzyme effectively removes branched chains during starch liquefaction,promoting further enzymatic hydrolysis.Although the exploration and application of SDBEs have been ongoing for many years,there remains a lack of understanding of the molecular mechanisms such as catalytic modes,thermal stability,and substrate specificity.This knowledge gap makes it challenging to enhance catalytic performance through molecular modification.In this study,a glycogen debranching enzyme gene from the thermophilic archaeon Saccharolobus solfataricus STB09（Ss GDE）was discovered.Biochemical analysis revealed that Ss GDE exhibited higher efficiency in debranching maltodextrin compared to amylopectin,potentially making it advantageous for application in starch saccharification.Furthermore,we explored and revealed the molecular mechanisms of thermal activation and substrate specificity of Ss GDE.The main research includes:（1）The successful construction of the Ssgde/p ET-20b（+）E.coli expression system has allowed for the production and extracellular secretion of recombinant Ss GDE.The influence of the histidine purification tag（His-tag）on the catalytic performance of recombinant Ss GDE was investigated.The shaking flask fermentation conditions for recombinant Ss GDE were optimized,including the identification of the optimal medium and fermentation conditions.The results indicated that adding His-tag at the N-terminus or C-terminus of recombinant Ss GDE significantly impaired its catalytic activity.The enzymatic activity was highest when the recombinant Ss GDE was devoid of any purification tag.The optimal culture medium for the production of recombinant Ss GDE is TB medium.When the p H of the medium is 7.0,the inducer is isopropyl-β-D-thiogalactopyranoside（IPTG）at a final concentration of 0.05 m M,and the fermentation temperature is 25°C,the highest level of extracellular production of recombinant Ss GDE is achieved.When fermented for 108 hours,the extracellular enzyme activity reached 1300 U/m L,as measured using the iodine colorimetric method.（2）To further investigate the catalytic characteristics of recombinant Ss GDE,purification was performed using anion exchange chromatography and hydrophobic affinity chromatography methods.Biochemical analysis revealed that the optimal temperature of the recombinant Ss GDE was 70°C.It had superior thermal stability and exhibited thermal activation characteristics.After incubation at 70°C for 4 h,the enzyme activity of recombinant Ss GDE increased by 126%compared to the initial.The optimal p H for recombinant Ss GDE was 5.0.Recombinant Ss GDE exhibited stability in neutral or slightly acidic（p H 5.0 to 7.0）environments.The activity of recombinant Ss GDE was inhibited by Fe³⁺ions,while it could be enhanced by Zn²⁺and Mg²⁺ions.The presence of Na Cl（200 to 600 m M）could also enhance the enzyme activity.The storage stability of recombinant Ss GDE was investigated,and the results indicated that it was suitable for storage under freezing conditions such as-80℃or-20℃.At 4℃and 30℃,the enzymatic activity of recombinant Ss GDE significantly decreased with increasing storage time.Recombinant Ss GDE could not hydrolyze theα-1,4 glycosidic bonds in starch substrates.It also had difficulty in acting on pullulan.Recombinant Ss GDE was capable of acting on amylopectin and maltodextrin.Dextrose equivalent 4（DE4）maltodextrin is the optimal substrate for recombinant Ss GDE.The hydrolytic activity measured using the3,5-dinitrosalicylic acid（DNS）method is 22 times higher than using corn amylopectin and potato amylopectin as substrates.When using high DE value（DE16 and DE19）maltodextrin as substrates,the hydrolytic activity of recombinant Ss GDE decreased to approximately 70%of that observed using the DE4 maltodextrin.Product analysis indicated that recombinant Ss GDE preferentially hydrolyzed short-branched chains with degree of polymerization（DP）between 5-10,followed by slower hydrolysis of long-branched chains（DP 11-20 and DP>21）.It had limited activity on branches with DP<5.（3）Ss GDE crystals were successfully obtained using protein crystallization techniques.The three-dimensional structure of Ss GDE was determined through X-ray diffraction.The obtained structure was submitted to the Protein Data Bank（PDB）and assigned the PDB ID:7EAV.Ss GDE contained three domains:domain N（residues 1-153）,domain A（residues 154-590）,and domain C（residues 591-718）.Among them,Asp363,Glu399,and Asp471 formed the catalytic triad.The crystal structure revealed that the region with high B-factor values in Ss GDE was primarily located in domain C.The average B-factor values for domain C in two subunits within one asymmetric unit cell were 70.21（?）² and 71.17（?）²,respectively,indicating a high degree of flexibility in this region.The low B-factor values were mainly distributed in the region where the two subunits contacted each other,indicating that dimerization contributed to the stability of the Ss GDE structure.In the Ss GDE structure,no Ca²⁺binding sites were found.Two subunits arranged in a head-to-tail manner were proposed for the dimeric form of Ss GDE due to the high interaction surface area and the strongest interaction force.（4）The correlation between the thermal activation characteristic of recombinant Ss GDE and its quaternary structure was investigated by exploring the molecular weight of recombinant Ss GDE under different temperatures（30℃and 70℃）.The results revealed that the peak time of recombinant Ss GDE incubated at 70℃was consistent with that of recombinant Ss GDE incubated at 30℃,corresponding to a molecular weight of approximately 136 k D.This indicated that the thermally treated Ss GDE remained in a dimeric form and did not undergo any transition to other oligomeric forms.Further investigation was carried out using molecular dynamics simulations to explore the changes in the quaternary structure of Ss GDE at different temperatures.At different simulation temperatures（30℃,50℃,and 70℃）,the gyration radius（R_g）values of the Ss GDE subunits remained at the same level,indicating the absence of folding and unfolding tendencies.The average R_g value of the Ss GDE dimer was found to be 37.3（?）,with small fluctuations.Increasing the simulation temperature did not lead to either aggregation or dissociation of the dimeric Ss GDE.Therefore,the thermal activation characteristics of Ss GDE may be independent of its quaternary structure.As the simulation temperature rose,the solvent-accessible surface area（SASA）of the catalytic cavity in Ss GDE also increased.This led to more amino acid residues being exposed to the surrounding solvent.An analysis was conducted on the conformational changes of four loop regions（loop1 313-337,loop2 399-418,loop3 481-513,and loop4 540-574）involved in the formation of the catalytic cavity in Ss GDE.The results indicated that an increase in simulation temperature prompted loop1,loop2,loop3,and loop4 to move away from the catalytic cavity,resulting in the expansion of the Ss GDE catalytic cavity.Within the catalytic cavity of Ss GDE,loop3 was notably longer than the corresponding loops in other GH13 family amylases.Additionally,the catalytic residue Asp471was situated beneath loop3.At low temperatures,loop2 and loop3 in Ss GDE were in close proximity,resulting in a narrower catalytic cavity compared to other GH13 family amylases.This proximity hindered the interaction between catalytic residues and substrate.However,after thermal incubation,the conformation of the catalytic cavity was modulated,exposing the catalytic amino acids.This increased exposure enhanced the frequency of interaction with the substrate,ultimately improving the catalytic performance of Ss GDE.（5）The molecular mechanism of substrate specificity in recombinant Ss GDE was investigated by comparing the binding modes of Ss GDE with two other enzymes,Pseudomonas amyloderamosa isoamylase（Pa ISO）and Chlamydomonas reinhardtii isoamylase（Cr ISO）,using molecular docking and molecular dynamics simulations.First,molecular docking was performed to dock maltopentaose（G5）and maltodecose（G10）into the catalytic cavity of the three SDBEs,resulting in the formation of complex structures including Ss GDE-G5,Ss GDE-G10,Pa ISO-G5,Pa ISO-G10,Cr ISO-G5,and Cr ISO-G10.The results indicated that G5molecules could bind in a linear conformation within the catalytic cavities of Ss GDE,Pa ISO,and Cr ISO,with the reducing end interacting with the catalytic residues.On the other hand,G10 molecules adopted a bent conformation when bound within the catalytic cavity,with the non-reducing end observed to be bent.This indicates that the catalytic cavities of the debranching enzymes are unable to accommodate long-chain substrates and implies the presence of other substrate-binding regions near the catalytic cavities.Molecular dynamics simulations were performed on the complexed structures,revealing that the G5 molecule in the Ss GDE-G5 complex is pulled away from the catalytic center by the loops 224-232,493-497,and 557-558 of Ss GDE.The instability of G5 in the catalytic cavity may be the reason why Ss GDE has difficulty recognizing branches with DP<5.The simulation results for Ss GDE-G10,Pa ISO-G10,and Cr ISO-G10 systems were different than those of Ss GDE-G5,Pa ISO-G5,and Cr ISO-G5 systems.In the Ss GDE-G10 system,aromatic amino acid residues Phe232,Tyr244,Tyr322,Tyr323,Phe326,Phe491 and Phe557 primarily contributed to the interactions with G10.These aromatic amino acids were distributed more concentratedly on both sides of the catalytic cavity.In Pa ISO and Cr ISO,the aromatic amino acids that form strong interactions with the G10 substrate were more dispersedly distributed,forming a large active site.However,most of the aromatic amino acid residues that could form strong interactions with the substrate were not conserved in Ss GDE.Based on structural alignment,site-directed mutations were introduced to the corresponding amino acid residues in Ss GDE.This led to the construction of Ss GDE mutants Y323F,L324Y,F326N,F326Y,V375P,V375F,and YLDF/FYDN（loop323-326 in Ss GDE replaced by loop335-338 in Pa ISO）to investigate the impact of modulating the substrate-binding region of Ss GDE on its substrate specificity.The results showed that the mutants Y323F and V375F exhibited significantly enhanced hydrolytic activity towards maize amylopectin,potato amylopectin,and DE4 maltodextrin.Moreover,the improvement in hydrolytic activity towards high molecular mass amylopectin by the mutants was significantly greater than that towards maltodextrin.Molecular docking and molecular dynamics simulations were performed on the mutants Y323F and V375F with the G10 substrate,which further confirmed that the mutated amino acid residues exhibited stronger binding affinity towards the substrate compared to the wild-type Ss GDE.The overall analysis indicates that the substrate preference of Ss GDE for maltodextrins is determined by the relatively smaller substrate-binding region around the catalytic cativity.