Mechanism Of The Function Integration For Multifunctional Amylase OPMA-N

Glycoside hydrolase family13（GH13） has a large number of members andwhich shows a special effect on the α-glycosidic linkages, and it also calledα-amylase family. They show different types of catalytic properties, includinghydrolysis or transglycosylation for the α-（1,4） and α-（1,6）-glycosidic linkages. The20subfamily of the GH13（neopullulanase） is capable of catalyzing all or two ofabove types of reactions, it is also though of as a multi-functional amylase andbecame a hotspot for researching in nowadays. We have reported that theoligosaccharide-producing multifunctional amylase-N （OPMA-N） is a novel type ofstarch degrading enzyme secreted from Bacillus sp. ZW2531-1, it can degradedstarch to maltose, maltotriose, isomaltotriose and isomaltotetraose via reactions ofhydrolysis and transglycosylation, the isomaltooligosaccharides is known as bifidusfactors and has a great economic and medicinal value.In recent years, with the deepening understanding of protein structureinformation, as well as the development of computer technology, the technology ofreasonable design for protein has a rapid development and application. Combinewith protein engineering, we will not only to get better protein （or enzyme） forapplication, but also can deeply study the intrinsic relationship between proteinstructure and function. In this study, we first built the initial structure model ofOPMA-N by homology modeling, and then used the energy optimization andmolecular dynamics simulation to optimize the initial model, at last, multipleassessment software were adopted to prove the rationality of OPMA-N model.Combined with the analysis for model, and the homologous sequence alignment ofthe GH13family, two important residues （Trp358, Val328） were chosen forsite-directed mutagenesis, and recombinant enzymes with multi-N-domain ormulti-catalytic domain were built to study.We first constructed all of the possible3D structures with20different aminoacids at this site by homology modeling. After careful analysis, taking into theaccount factors such as sterics, polarity, and charge, the seven mutants W358K, W358R, W358N,W358T, W358G, W358D, andW358E were selected to representthe diversity of this site. After constructed mutant plasmids, we got pure enzymesafter expression and purification, and then their nature were determined, we foundthat when W mutated into a positively charged side chain of K/R, their productswere given priority to glucose, the ratio reached to83.6%and80.2%, at the sametime with a little maltose, while the products of wild type and other mutants had noglucose, the results show that W358K and W358R had lost most of thetransglycosylation activity, and became the starch hydrolyzing enzymes. In contrast,isomaltooligosaccharides in the products of W358N/T, W358D/E had increased, weillustrated that their transglycosylation activity had improved. Another significantchange was the activities of W358K/R had increased nearly40%, their Km valueshad decreased while their kcat values had increased compared to the wild type,mutation like them might increased affinity between enzyme with substrate and theircatalytic efficiencies. The enzyme activities of W358D/E were half higher than thewild type, while the Km and kcat values were increased, indicating that mutationslike them could improved catalytic efficiencies but also reduced the binding abilitybetween enzyme with substrate. The numerical of W358N/T were similar to the wildtype, while the W358G was far lower than that of the wild type OPMA-N incatalytic efficiency. The thermal stability of mutants W358K/R and W358D/E wasslightly better than the wild-type, we inferred that the introduction of the chargedand sterically hindered amino acid residues might increased the interaction with theiraround residues. Oligomerization results showed that different mutants, formeddifferent oligomerization which from monomer to pentamer, it （especially the ratioof monomer and dimer, amount of monomer and dimmer） might influenced thecatalytic properties of the enzyme molecules form and nature. Finally, through thecomputer simulation of molecular docking results, we wanted to find the molecularmechanism of the changes in enzymes’catalytic properties, wild type Trp358locatedin the substrate binding pocket+2sub-site and it formed very strong steric hindrance,this structure formed the activity balance between hydrolysis and transglycosylationfor OPMA-N, and it had changed in the mutants, there were newly formed hydrogen bonds between Asn/Thr358and Asp/Glu358with+3/+2sub-sites of substraterespectively, their interactions between them with substrate and Glu356had greatchanges. The strong interactions were formed between Lys/Arg358with substrateand catalytic residue Glu356. Mutant Gly358had lost the original steric interactionand the interaction with the surrounding residues.Val328was another important amino acids the mutation target that selected,according to research reported at the same time, we wanted to explore whether thesize of the residue’s side chain determines the catalytic type of multifunctionalamylase OPMA-N in this site, so three mutants V328I, V328A and V328G weredesigned respectively. Results showed that, in this site the bigger side chain of Ileand smaller side chains of Gly significantly decreased catalytic activity. When Valmutated into Ile, its product of isomaltooligosaccharides was significantly decreased,whereas the contents of V328A and V328G had increased slightly. Anothersignificant change was this enzyme had certain activity for β-CD when the mutationof Val into Ala, its products were mainly maltose and maltotriose. The mutation ofthis site had little effect on pH, temperature properties of enzymes, but influencedthe degree of oligomerization. Molecular docking results showed this site had acertain effect on the adjacent Asn330, receptor sugar binding site Glu331and theposition and orientation of the catalytic residues of Asp327, especially there weretwo newly hydrogen bonds were formed between Asn330with the+2sub-site ofsubstrate in mutant V328A, the bonds obviously pulled the substrate to the receptorsugar binding pocket direction, perhaps this was the reason of the changes in itssubstrate specificity and the product ratios.This article, on the other hand, focuses on the core of N-domain of theGH13₂0subfamily, the reconstruction vectors OPMA-NN, OPMA-TNT,OPMA-NTN were constructed, together with the original OPMA-N and OPMA-NT,the study on enzymatic properties detection of different recombinants and differentmodules combinations were found that their product proportion did not changesignificantly, the activities between the combination module and those beforecombination had littler difference, the activity of OPMA-TNT was slightly higher than that of the wild type. An obvious result was that the thermal stability ofrecombinant OPMA-NTN with two N-domain was increased by20℃comparedwith the wild type, combined with OPMA-N and OPMA-NT, we demonstrated thatN-domain had a great contribution for the thermal stability of OPMA-N.In conclusion, through the rationality mutation study of multifunctional amylaseOPMA-N, and the restructuring module, combined with molecular docking results,to a certain extent, revealed the mechanism of the function integration and catalyticactivity for OPMA-N. At the same time, we got some mutant enzymes such asOPMA-N/W358N, OPMA-N/V328A, OPMA-NTN which shown potential value inindustrial application.