Structure/Function Relationships And Molecular Modification Of Thermostable Amylosucrase

Carbohydrate-Active en Zymes（CAZymes）catalyze the assembly,cleavage,and modification of carbohydrates.Glycoside hydrolases（GHs）are the largest family of CAZymes and carry out carbohydrate degradation via hydrolysis and carbohydrate synthesis via transglycosylation.Application of GHs enables use of inexpensive biomass such as starches and sucrose as donor substrates in the synthesis of valuable biomolecules.Amylosucrase（AS,EC 2.4.1.4）is a multifunctional enzyme belonging to the GH13 family,and can catalyze several types of reactions including polymerization,isomerization,and transglycosylation.ASs can carry out these reactions using sucrose as the donor substrate.ASs have broad potential in the food industry for producing functional sweeteners,dietary fibers,carbohydrate-based carrier materials,and bioactive compounds.However,of the limited number of ASs identified,most show poor thermostability and inactivate rapidly at industrially relevant temperatures,severely limiting their applications.Recent studies have shown that most thermophilic proteins have a higher oligomerization state than their mesophilic homologs.The inter-subunit interaction of these oligomers is considered one of the primary mechanisms by which these thermophilic proteins are stabilized.Although the crystal structures of three ASs and their catalytic mechanism have been investigated,the interplay between their oligomerization state and thermal stability is less well understood.While it is known that dimeric AS has better thermal stability than its monomeric counterpart it is still poorly characterized,and attempts at improving thermostability have thus far not involved manipulating the quaternary structure.To address this,we mined the NCBI sequence database for a thermostable AS based on the features of its subunit interface,solved its crystal structure,and then investigated the roles of the interfacial residues through truncation and substitution mutations.Finally,we applied computer-aided rational design to strengthen the relatively weak subunit interface and improve the thermal stability of this AS.The main results are listed as follows.（1）Characterization of thermostable tetrameric CT-AS.Based on the long loop interface region of a dimeric AS,a putative AS（CT-AS）from the thermophilic microorganism Calidithermus timidus DSM 17022 was selected,its coding region was cloned to the p ET22b（+）vector,and then it was transformed into E.coli BL21（DE3）for heterologous expression and affinity purification.Characterization of the enzyme showed a p H and temperature optima of 7.0 and 55°C respectively.When sucrose is used as the sole substrate,CT-AS primarily catalyzes the polymerization of glucose to generateα-1,4-glucan.Addition of fructose as an acceptor drives the isomerization reaction to generate turanose.Size exclusion chromatography and static light scattering analysis show that CT-AS exists as a tetramer in solution.This is the first reported tetrameric AS.CT-AS is the most thermostable AS known with a T_m value of74.47°C and a t_1/2（60°C）of 46.21 h.（2）Crystal structure of tetrameric CT-AS.The three-dimensional structure of CT-ASase was solved at a resolution of 2.29?.Each asymmetric unit in the crystal structure of CT-AS is composed of four subunits.There are two kinds of subunit interface:the intra-dimer interface（which also exists in the dimeric AS）,and the inter-dimer interface which is unique to this tetrameric AS.The inter-dimer interface shows shape complementarity,with the main interactions being hydrophobic interactions and hydrogen bonding.The buried area of the inter-dimer interface is about half of that of the intra-dimer interface.The quaternary structure of tetrameric CT-AS is different from that of other tetrameric proteins of the GH13 family,revealing an unreported subunit assembly pattern within this family.（3）The effect of interface residues on the thermal stability of CT-AS by site-directed mutagenesis.Truncation and substitution mutations were introduced into the two long loop regions of the intra-dimer interface.These mutations revealed that these long loop regions impact thermal stability.The T_m values of these mutants decreased by up to 14.79°C,but their oligomeric states in solution were not changed.Alanine scanning was carried out on the five residues forming hydrogen bonds in the inter-dimer interface.The T_m values of these mutants decreased by up to 5.50°C.Moreover,the mutant harbouring 5 mutation sites and the mutant N577A exist as a monomer in the solution,indicating that the disassembly of the inter-dimer interface will affect the oligomerization state of the protein.At the same time,while the thermal stability of CT-AS cannot be easily improved by only optimizing residue 577,such work highlights that the interactions of inter-dimer interface are relatively weak and could potentially be stabilized by other mutations.（4）Rational design to improve the thermal stability of CT-AS.The rational design used to improve the thermostability of CT-AS utilized five common strategies:interface engineering,folding energy calculations,consensus sequence analysis,hydrophobic effects enhancement,and B-factor analysis.By combining these techniques twelve candidate mutants were obtained;five of which had improved T_m value.Subsequent analysis of the homology model of the best single mutant H631R showed that this mutation generated two new hydrogen bonds at the inter-dimer interface.Then,these five mutations were combined.From this a further improved mutant M31（H631R/L382P/S414N/P618I）with even higher thermal stability was obtained.Of note,the t_1/2 at 65°C increased by 20.02 h compared to the wild type.The yield and chain length ofα-glucan synthesized using M31 at 50°C were higher than that of using the wild type,perhaps because these mutation sites were selected by multiple strategies and were located far from the active sites.Molecular dynamics simulations further indicated that strengthening the interaction network of the relatively weak inter-dimer interface can considerably impact thermostability.Taken together,this protein engineering campaign has yielded a far improved mutant with higher activity at industrially relevant temperatures compared to any known AS.