Selective Mechanism Of The Hydrolysis And Transglycosylation Of α-glycosidases

Since glycosidases（GHs）can catalyze the hydrolysis or transglycosylation reactions of glycosides independently of’activated’sugar phosphates,they have the potential to facilitate in vitro glycoside synthesis through transglycosylation.However,many GHs exhibit both hydrolysis and transglycosylation activities,leading to lower product yields of glycosides.Therefore,understanding the selective mechanism of hydrolysis and transglycosylation reactions in GHs is crucial for achieving directional transglycosylation and improving glycoside synthesis.Moreover,the type of glycosidic bond significantly influences the structure and function of glycosides,underscoring the importance of linkage specificity in transglycosylation reactions.During hydrolysis or transglycosylation catalyzed by GHs,a transition state（TS）structure is formed,and the free energy barrier of this structure determines the preference of GHs for hydrolysis or transglycosylation.However,capturing the TS structure experimentally is challenging and represents a critical aspect in studying the mechanism of hydrolysis and transglycosylation.Multi-scale theoretical simulations can be used to model the the catalytic mechanism of GHs and obtain key TS structures.Whileβ-glucosidases have been extensively studied,the mechanism ofα-glucosidases,widely used in the field of food processing,remains less explored.This research utilizes a multi-scale theoretical simulation approach to investigate the hydrolysis preference mechanism of fucosidase from Lactobacillus casei Alf C in the GH29family and the transglycosylation preference mechanism of amylomaltase from Thermus aquaticus Ta AM in the GH77 family.Based on the distribution characteristics of key sites influencing hydrolysis and transglycosylation preferences,molecular modifications were performed on sucrose hydrolases and amylosucrases to achieve directional regulation of hydrolysis and transglycosylation.The selective mechanisms were deciphered using multiscale theoretical simulations.Furthermore,the mechanism underlying the preference for transglycosidic bond type in the 4,6-α-glucanotransferase from Limosilactobacillus reuteri 121Lr Gtf B was revealed.The main findings are outlined below:（1）The mechanism of hydrolysis preference in the GH29 family fucosidase Alf C was investigated.To address the incorrect positioning of acid/base catalysts,accelerated molecular dynamics simulations were employed to sample the conformation of the loop containing the acid/base candidate residue D242.D242 was identified as the acid/base catalyst,and its catalytic conformation was determined.QM/MM metadynamics simulations revealed free energy barriers of 16.1 kcal·mol^-1,9.8 kcal·mol^-1 and 11.4 kcal·mol^-1,respectively.Alf C exhibited a preference for hydrolysis.The conformational pathways of the three reactions were determined as ¹C₄→[³H₄]^?→¹C₄,³S₁/³H₄→[³H₄/E₄]^?→¹C₄ and ³S₁/³H₄→[E₄]^?→¹C₄,respectively.Analysis of the TS structures for hydrolysis and transglycosylation reactions revealed electrostatic repulsion between the carboxyl O_δ2 atom of D242 and the O5 atom of the acceptor sugar ring in the transglycosylation TS structure.This repulsion contributed to a higher free energy barrier for transglycosylation,explaining the hydrolysis preference in Alf C.（2）The mechanism of transglycosidic preference in the GH77 family amylomaltase Ta AM was investigated.To address the issue of the sugar chain being distant from the acid/base residue E340 and unable to complete proton transfer,a loop-mediated conformational switch model involving sugar chain sliding was proposed.Path-metadynamics simulations demonstrated that the presence or absence of sugar chains corresponded to closed（C）and open（O）conformations of the 250s loop,respectively,with the C conformation being energetically higher by 4.0kcal·mol^-1.In the C conformation,the sugar chain could induce sliding,bringing the glycosidic oxygen atom close to the acid/base residue E340 to catalyze the glycosylation reaction with a free energy barrier of 12.9 kcal·mol^-1.Path-metadynamics results indicated that the 250s loop adopted a partially closed（PC）conformation during hydrolysis and a C conformation during transglycosylation.QM/MM metadynamics simulations revealed free energy barriers of 18.0kcal·mol^-1 and 9.0 kcal·mol^-1for hydrolysis and transglycosylation reactions,respectively.The energy barrier for transglycosylation was 5.1 kcal·mol^-1 lower than that of hydrolysis,consistent with the experimental ratio of 5000:1 for transglycosylation/hydrolysis.Superimposition of the TS structures of hydrolysis and transglycosylation reactions revealed that the C state of the 250s loop during transglycosylation resulted in a more stable TS,while the PC state of the 250s loop during hydrolysis led to an unstable TS.Therefore,the difference in transition state energy induced by conformational dynamics was identified as a crucial factor for Ta AM’s preference for transglycosidic reactions,which was further supported by experimental findings of reduced disproportionation/hydrolysis ratios upon mutations near the 250s loop.（3）Directed regulation of hydrolysis and transglycosylation in sucrose hydrolases（SHs）and amylosucrases（ASs）of the GH13 family was explored.SHs favor hydrolysis,while ASs prefer transglycosylation.Sequence alignment and structural analysis revealed conserved amino acids S（located in SHs）and A（located in ASs）adjacent to the nucleophilic residue D.Site-directed mutagenesis of Xa SH（Xanthomonas axonopodus）,Cc SH（Caulobacter crescentus）,Np AS（Neisseria polysaccharea）,and Dg AS（Deinococcus geothermalis）was performed.The T/H（transglycosylation/hydrolysis）ratios of Xa SH_S281A and Cc SH_S271Aincreased from 0.05 and 0.07 of the wild type to 1.4 and 1.11,respectively,while the T/H ratios of Np AS_A287S and Dg AS_A285S decreased from 8.80 and 7.13 of the wild type to 0.15 and 0.20.Enzyme kinetic data,HPLC,HPAEC-PAD,and ¹H NMR results supported the switch in preference for Xa SH_S281A and Cc SH_S271A from hydrolysis to transglycosidic reactions and for Np AS_A287Sand Dg AS_A285S from transglycosidic to hydrolysis reactions.（4）The mechanisms underlying the hydrolysis and transglycosylation selectivity of SHs and ASs in the GH13 family were investigated using Xa SH,Np AS,and their mutants.Molecular dynamics simulations revealed hydrogen bonds between S of Xa SH and Np AS_A287Swith the nucleophilic residue D,inducing a shift in the acceptor sugar molecule’s position and facilitating water penetration for hydrolysis.This hydrogen bond also decreased the p K_a value of the nucleophile,aiding glycosidic bond cleavage during deglycosylation.Free energy barriers for hydrolysis reactions of Xa SH,Xa SH_S281A,Np AS,and Np AS_A287S were determined as 10.3,13.5,8.9,and 7.1 kcal·mol^-1,respectively.Transglycosylation barriers were 20.3,9.6,8.2,and 9.6 kcal·mol^-1,consistent with experimental results.Analysis of the TS structure of transglycosylation revealed that the presence of a hydrogen bond induced a conformational shift in the sugar ring at the+1 subsite,resulting in steric hindrance with the sugar ring at the-1subsite,leading to a higher energy barrier for transglycosylation,explaining SHs’hydrolysis preference.Conversely,the absence of this hydrogen bond eliminated the steric hindrance,favoring in ASs.The steric hindrance effect induced by hydrogen bonding is the key factor for the preference of ASs/SHs for transglycosylation/hydrolysis.（5）Mechanism ofα-1,6/α-1,4 bond-type selectivity for Lr Gtf B of the GH70 family were explored.QM/MM metadynamics simulations showed that the glycosylation reaction of Lr Gtf B has an energy barrier of 12.2 kcal·mol^-1.The energy barriers forα-1,6 andα-1,4transglycosylation reactions were 6.7 kcal·mol^-1 and 22.3 kcal·mol^-1,respectively,when isomaltose was used as the acceptor.When maltose was used as the acceptor,the energy barriers forα-1,6 andα-1,4 transglycosylation reactions were 11.7 kcal·mol^-1 and 14.8 kcal·mol^-1.These findings establish Lr Gtf B’s strong preference forα-1,6 transglycosylation.Analysis of the TS structure during transglycosylation revealed the influence of the conformation of loop B located above the+1 subsite.The presence of the sugar induced plasticity in loop B duringα-1,6transglycosylation,resulting in a specific conformational change.Notably,the hydrophobic methyl group of T920 in loop B shifted towards the+1 subsite,thereby stabilizing the sugar ring conformation and promoting theα-1,6 transglycosylation reaction.In the case ofα-1,4transglycosylation,a hydrogen bond formed between K1128 and the sugar unit at the+1 subsite.This interaction constrained the conformation of sugar at the+1 subsite,leading to steric hindrance between the sugar at the-1 subsite and the sugar unit at the+1 subsite in the TS structure,enabling it to favorα-1,6 transglycosylation overα-1,4 transglycosylation.