Study On The Mining And Catalytic Properties Of Conserved Residues In Glycoside Hydrolase 13 Family And Its Subfamily

Conserved residues refer to highly conserved amino acid residues within multiple members of an enzyme family or the same functional category.These residues remain relatively fixed in the enzyme structure,maintaining consistent amino acid types and physicochemical properties.Conserved residues not only participate in substrate recognition and catalytic reactions but also play a crucial role insustaining enzyme catalytic activity and substrate specificity.The Glycoside hydrolase 13 family(GH13)encompasses diverse amylolytic enzymes with different catalytic properties,including α-amylases(AMY),α-glucosidases(AGL),oligo-1,6-glucosidases(OG),and glycogen branching enzymes(GBE).Amylolytic enzymes within this family are further categorized into different subfamilies based on their catalytic characteristics.Despite the recognized involvement of conserved residues in regulating enzyme catalytic properties,the specific conserved residues at the GH13 family and subfamily levels remain undisclosed.Additionally,the manner in which these residues regulate the catalytic properties of amylolytic enzymes within the family and its subfamilies remains unclear.Therefore,in this study,we focused on amylolytic enzymes of the GH13 family and its subfamilies to uncover conserved residues beyond the catalytic residues.Our analysis delved into the structural features of conserved residues within the GH13 family and its subfamilies,and elucidated their roles in enzyme catalysis.Furthermore,to investigate the relationship between conserved residues and catalytic properties,we examined the conserved residues associated with catalytic characteristics within specific subfamilies and studied their structural features and mechanisms in regulating substrate recognition and specificity.The main results are as follows:(1)Sequences analysis of the GH13 family and its subfamilies was performed.The results revealed an overall sequence conservation ranging from 2.3% to 3.5% within the GH13 family.Within the subfamilies,significant variations in sequence conservation were observed within the GH13 subfamilies due to specificity differences among enzymes,ranging from 30% for GH13＿33 to 80% for GH13＿1.The analysis of sequence conservation within the GH13 family and its subfamilies identified several conserved residues,including G37,P45,R52,Y57,D101,V103,H106,G230,R232,D234,E264,H330,D331,and G360(BsMFA numbering).The number of conserved amino acids varied from 15 to 266 within the subfamilies.(2)Structure and functional characteristics of conserved residues in the GH13 family were investigated with reference to the primary and crystal structure of BsMFA(PDB ID:6AG0).Analysis of the primary structure of BsMFA revealed that 12 conserved residues are located within the conserved sequence region(CSR)of the GH13 family.Crystal structure analysis of BsMFA demonstrated that the conserved residues of the GH13 family are situated within the substrate-binding pocket of the(β/α)8 catalytic barrel structure.Mutants of the GH13 family with mutations in conserved residues,especially those within the CSR,showed a significant decrease in enzyme activity,and in some cases,the activity was completely abolished.(3)The GH13＿5 subfamily,containing AMY with favorable substrate specificity,was selected for in-depth analysis to investigate the structural characteristics and functional implications of conserved residues within this subfamily.Conserved residues in the GH13＿5subfamily were identified as L65,E67,F68,D111,E114,R126,R147,F154,W156,F161,G163,D165,W218,V342,W345,and F346(BsMFA numbering).Structural analysis revealed that conserved residues L65,E67,F68,D111,E114,R126,R147,F154,W156,F161,G163,D165,and W218 are distributed within the cleft formed by structural domains A and B.Conserved residues V342,W345,and F346 are located within the cleft formed by structural domains A and C.The correlated motions of structural domains suggested significant flexibility in residue segments 104-114,118-137,140-157,165-177,180-184,and 189-194 in domain B.Among these segments,104-114,140-157,and 189-194 exhibited a negative correlation with the motion pattern of domain B but a synergistic trend with the motion pattern of domain A.The regular motion of these residue segments aids in identifying the sugar units of substrates and selecting specific substrates.Furthermore,structural analysis revealed that domain C,along with the disordered loop structure from F326 to W345,constituted a delivery channel from domain C to the substrate-binding pocket.Conserved residues V342,W345,and F346 within this channel indicated their involvement in substrate binding,recognition,and transportation within this subfamily.(4)To explore the conserved residues associated with the recognition,formation,or cleavage of α-1,6-glycosidic bonds,E148 and W198 were identified as key regulatory sites targeting the α-1,6-glycosidic bond through Shannon entropy analysis.The crystal structure(PDB ID: 6JOY)revealed that the conserved residues E148 and W198 are located within the substrate-binding pocket.E148 is situated in a β-fold region of the(β/α)8 catalytic barrel structure,with a very low B factor,and the distance between E148 and the substrate is greater than 6 (?).On the other hand,W198 is positioned within the irregular loop structure between highly fluctuating α7 and α8 helices,with the distance to the substrate less than 4 (?).Mutations were introduced to the conserved residues E148 and W198,and the enzymatic activity,molecular weight distribution,chain length distribution,and iodine-binding capacity of the mutant variants were assessed to determine the functional roles of these conserved residues in α-1,6-glucanase.The results demonstrated that mutations in the conserved residues led to a significant reduction in transglycosylation activity,with activity completely lost in some cases,while the impact on hydrolytic activity was minimal.Structural analysis of the product revealed a sharp decrease in the ability of the mutants to catalyze the formation of branched polysaccharides.(5)To elucidate the catalytic mechanism of the enzyme in hydrolyzing different types of glycosidic bonds,the subfamily GH13＿31 with dual specificity was selected to explore substrate-specific regulatory residues.Within the GH13＿31 subfamily,conserved residues that appeared regularly depending on the catalyzed glycosidic bond type were identified,namely residue V219 adjacent to catalytic residue D218 in CSR II,residue P275 in CSR III,and residue N344 in CSR IV(psp OG numbering).Mutant variants were constructed for the above residues,and it was found that the V219 A and V219A/N344 E variants could hydrolyze bothα-1,4-glycosidic bonds in G3 and α-1,6-glycosidic bonds in IG3,although weaker activity was observed in the V219A/N344 E variant.The V219A/P275 N variant exhibited a shift in substrate specificity from catalyzing α-1,6-glycosidic bonds in IG3 to catalyzing α-1,4-glycosidic bonds in G3.To evaluate the practical value of psp OG and its variants,the G1 content was analyzed as an indicator,specifically the increase in G1 from a 10% glucose mother liquor substrate at 50°C.The V219 A mutation increased the proportion of G1 in the first,liquor,second,and third mother liquors from the original 91.09%,76.59%,and 47.53%to 96.88%,95.70%,and 90.46%,respectively.The mixture of V219 A and V219A/P275 N showed the proportions of G1 in the first,second,and third mother liquors increasing from the original 91.09%,76.59%,and 47.53% to 96.86%,93.63%,and 89.27%,respectively.Conserved residues are amino acid residues that remain highly conserved thorughout the process of evolution,playing a crucial role in the catalytic mechanism and substrate specificity of enzymes.Exploring conserved residues at different levels contributes to uncovering more details about catalytic mechanism and substrate specificity of enzymes.This exploration aids in the design and optimization of enzyme catalytic performance,further advancing the fields of biocatalysis and enzyme engineering.Furthermore,deepening our understanding of conserved residues enables the development of new tools and strategies to address complex catalytic challenges,thereby promoting the process of biomanufacturing and green chemistry.