?/?-N-Acetylhexosaminidase:Gene Mining,Molecular Evolution And Transglycosylation

GalNAc-and GlcNAc-containing oligosaccharides are widely distributed in microorganisms,plants,and animals,and they play important biological roles.They are present in sugar structures of gangliosides,N-glycan,mucin-type O-glycan,blood group antigens,tumor-associated glycans,milk oligosaccharides,and chitin.?-N-Acetylhexosaminyl lactose(HexNAc-Lac)is a class of important oligosaccharides.GalNAc?1-3Lac shares oligosaccharide constituents with the blood group P-related glycan,which inhibits respiratory pathogens.GlcNAc?1-3Lac is the sugar unit of many cancer-related carbohydrate antigens,such as KH-1 adenocarcinoma antigen,N3 antigens associated with gastrointestinal cancer,and primary acute myeloid leukemia antigen,which may serve as a potential biomarker and a target for therapy.However,their use is limited because of the difficulty of large-scale production of oligosaccharides.?-N-Acetylhexosaminidases(EC 3.2.1.52)are an important class of glycosidases that catalyze hydrolysis of terminal N-acetyl-?-D-hexosamine(HexNAc),N-acetyl-?-D-galactosamine(GalNAc),or N-acetyl-?-D-glucosamine(GlcNAc)from various oligosaccharides and glycoconjugates.Based on amino acid homology,?-N-acetylhexosaminidases are classified into glycoside hydrolase(GH)families 3,20,84,and 123.These enzymes are widely distributed in microorganisms,plants,and animals,and they play crucial roles in nature.For instance,they can cleave a GalNAc residue from GM2 gangliosidose,and their deficiency will cause lysosomal storage diseases.They are also involved in bacterial cell wall recycling,fungal cell wall chitin lysis,and the insect life cycle.Some kinds of ?-N-acetylhexosaminidases are known to perform transglycosylation reactions,which can transfer N-acetylaminoshexosyl catalyze glycosyl transfer to form ?-N-acetyl-D-hexosaminide.Multiple ?-N-acetylhexosaminidases can be produced for utilization of chitin,intestinal mucosal glycans,and milk oligosaccharides by some intestinal bacteria,such as Bacteroides fragilis,Clostridium perfringens,and Bifidobacterium bifidum.Genes of 13 ?-N-acetylhexosaminidases belonging to GH20,GH84,and GH123 were cloned from B.fragilis ATCC 25285,C.perfringens ATCC 13124,and B.bifidum JCM 1254 and then successfully expressed in E.coli.The recombinant enzymes were purified and incubated with pNP-?-HexNAc as a glycosyl donor and lactose as an acceptor to detect transglycosylation activity.The results showed that 11 enzymes exhibited transglycosylation activities.Interestingly,BbhI from B.bifidum JCM 1254,an enzyme in the GH20 family,exhibited strict regioselectivity toward lactose without isomer production from both glycosyl donors.Bbhl also exhibited the highest transglycosylation activity regardless of the kind of glycosyl donor.The enzymatic properties of ?-N-acetylhexosaminidase BbhI were studied in detail and the synthesis conditions were optimized to improve the yield.With respect to substrate specificity for hydrolysis,BbhI was active when pNP-?-GalNAc or pNP-?-GlcNAc was used.The result showed that no activity existed toward the other substrates with ?-linkages or without GalNAc or GlcNAc in the glycon moieties.Furthermore,Bbhl was a non-ionic-dependent glycosidase.The enzyme was highly active at pH 5.0 to 6.0 and stable at pH 4.0 to 11.0.Bbhl showed an optimal temperature at approximately 45? to 55?,and it was stable below 55?.This enzyme possesses the bifunctional property of efficiently transferring both GalNAc and GlcNAc residues through ?1-3 linkage to the Gal residue of lactose.The effects of initial substrate concentration,pH,temperature,and reaction time on transglycosylation activities of BbhI were studied in detail.The enzyme was used at concentrations of 2 mg/mL(0.06 U/mL)for pNP-?-GalNAc and 0.02 mg/mL(0.12 U/mL)for pNP-?-GlcNAc.With the use of 10 mM pNP-?-GalNAc or 20 mM pNP-?-GlcNAc as the donor and 400 mM lactose as the acceptor in phosphate buffer(pH 5.8),BbhI synthesized GalNAc?1-3Galpl-4Glc and GlcNAc?1-3Gal?1-4Glc at maximal yields of 55.4%at 45? and 4 h and 44.9%at 55? and 1.5 h,respectively.The model docking of BbhI with lactose showed the possible molecular basis of strict regioselectivity of ?1-3 linkage in ?-N-acetylhexosaminyl lactose synthesis.Transglycosylation products could be formed by Bbhl in the reactions with pNP-?-GalNAc or pNP-?-GlcNAc as donor and maltose,sucrose,lactose,methyl ?-lactosid,galactose,N-acetyl-?-D-galactosamine,rhamnose,arabinose,methanol,ethanol and n-propanol as acceptor.The transglycosylation catalyzed by ?-N-acetylhexosaminidase proceeds through the same reaction mechanism as general glycosidases,resulting in moderate product yields.Although the yield of glycoside products can be improved by optimizing the reaction conditions,it only promotes the reaction equilibrium to the glycosidic bond synthesis in a certain extent.Therefore,in order to obtain higher transglycosylation activity of ?-N-acetylhexosaminidase,mutagenesis of BbhI was carried out.Random mutagenesis is an effective way for molecular modification of glycosidases.Traditional random mutagenesis is a full-length error-prone PCR of the target gene.The PCR products are digested and ligated after being recovered,and then transformed.This method has heavy screening work.The low efficiency of ligation leads to the loss of some error-prone PCR products.The random mutagenesis of megaprimer can randomly mutate specific target regions which only need two-step PCR.We first randomized the domain sequence by error-prone PCR and the resultant fragment was used as a megaprimer that replaces a homologous region in the template plasmid.The mixture was treated with DpnI to digest the template plasmid.The DpnI-treated mixture was then introduced into competent E.coli cells to yield plasmids carrying replaced insert fragments.There was no self-association of the plasmid.Compared with the traditional method,the operation steps and cost were reduced,but the efficiency was higher.In this paper,the BbhI was subjected to random mutagenesis of a specific region by MEG A WHOP(megaprimer PCR of whole plasmid).At present,there is no report about the enhancement of ?-N-acetylhexosaminidase transglycosylation efficiency by random mutation.Three mutants RMe4,RM34 and RMf125 that exhibited elevated transglycosylation activity compared with BbhI were selected.Among them,nine mutant sites N468I,M515K,Y562F,N649S,K653R,N670Y,I684A,D714G and S828I were present in RM34.Sequencing results from RMe4 indicated the following point mutations N468S,I478N,N521S,W773R and K420M.RMf125 sequencing results showed I453F,D714V,I730T,E755A and S857G.RM34 and RMf125 contained the same mutation site D714,suggesting that D714 plays an important role in transglycosylation.Both D714 and W773 played a key role in the increase of product yield.D714 was found to mutate aspartic acid to threonine(D714T)at position 714,and the yield of product was increased from 44%to 84%of the wild-type enzyme(44%).W773R exhibited elevated product yield from 44%to 81%of the wild-type enzyme(44%).The kinetic parameters of pNP-?-GlcNAc were compared with the mutant enzymes D714T and W773R.The results showed that the Km values of mutant D714T and W773R were lower than that of the wild-type enzyme,while the kcat value was 206 and 17 times lower than that of the wild-type enzyme,respectively.By comparing the mutant enzymes with the kinetic parameters of wild-type enzyme,it was found that the mutant enzyme caused a decrease in the affinity(increased Km)of the enzyme to the substrate and the strong decrease in the catalytic ability(reduced kcat)of the enzyme,which lead to a lower hydrolysis(kcat/Km).The structure of the wild-type enzyme and the mutant D714T and W773R were analyzed by bioinformatics,and the structural basis of the improved transglycosylation efficiency was explored.The model comparisons and collective motion analysis showed that the catalytic cavities of mutant D714T and W773R did not change.This results are also verified by the NMR data.However,the mutation of D714T and W773R resulted in a large change in the external structure of the catalytic chamber.After the mutation of nucleophile assistant residue D714,the yield of product increased greatly,and the mutant enzyme had no ability to hydrolyze the product.Since arginine was more likely to react with water molecule with H-bond,W773R could capture water molecules outside the catalytic cavity to reduce water activity thus improve the transglycosylation activity.Mucin-type oligosaccharides(O-glycans)are involved in several important biological events including cell-to-cell communication in higher eukaryotes,bacterial adhesion to host cells,and so on.?-GalNAc is the basic linking unit in most O-glycoproteins,where it is attached as the first carbohydrate unit to serine or threonine(Tn antigen).An abnormal mucin-type O-glycan whose expression is associated with cancer and several human disorders is the Tn antigen.Moreover,GalNAc linked to Ser/Thr is modified by the addition of sugars such as Gal,GalNAc,and GlcNAc to form eight different types of core structures.Among them galactosyl?1-3-N-acetyl-D-galactosamine(Gal?1-3GalNAc)attached to the serine or threonine residue via an a-linkage(Core 1,also referred to as T antigen)is one of the most abundant core structures found in mucin glycoproteins that are widely distributed in intestinal tracts of human and animals.However,it has been shown that T antigen is frequently exposed on the cell surface of carcinoma and T-cell lymphoma,and this unmasked form is thought to be involved in tumor cell adhesion and tissue invasion.?-N-Acetylgalactosaminidases(EC 3.2.1.49)are an important class of glycosidases that catalyze hydrolysis of terminal N-acetyl-a-D-galactosamine(GalNAc)from various oligosaccharides and glycoconjugates.Based on amino acid homology,?-N-acetylgalactosaminidases are classified into glycoside hydrolase(GH)families 27,36,109,and 129.These enzymes are widely distributed in microorganisms,plants,and animals,and they play crucial roles in nature.Some kinds of ?-N-acetylgalactosaminidases are known to perform transglycosylation reactions,which can catalyze glycosyl transfer to form ?-N-acetyl-D-galactosaminide.Multiple ?-N-acetylgalactosaminidases can be produced for utilization of O-glycan by some intestinal bacteria,such as Bacteroides fragilis,Clostridium perfringens,and Bifidobacterium longum.In this paper,Genes of 3 ?-N-acetylgalactosaminidases belonging to GH36,GH109,and GH129 were cloned from B.fragilis ATCC 25285,C.perfringens ATCC 13124,and B.longum JCM 1217 and then successfully expressed in E.coli.The recombinant enzymes were purified and incubated with substrats to detect transglycosylation activity.The results showed that 2 enzymes exhibited ?-N-acetylgalactosaminidase activities,but not BF0874,obtained from B.fragilis ATCC 25285.Interestingly,BLLJ0788 from B.longum JCM 1217 can use GalNAc-?-pNP or Gal?1-3GalNAc-?-pNP as a donor and Ser or Thr as the receptor for transglycosylation.The enzymatic properties of ?-N-acetylgalactosaminidase BLLJ0788 were studied in detail and the synthesis conditions were optimized to improve the yield.With respect to substrate specificity for hydrolysis,BLLJ0788 was active when GalNAc-?-pNP or Gal?1-3GalNAc-?-pNP was used.The result showed that no activity existed toward the other substrates with ?-linkages or without GalNAc in the glycon moieties.Furthermore,BLLJ0788 was a non-ionic-dependent glycosidase.The enzyme showed an optimal temperature at approximately 55? to 60?,and it was stable below 50?.This enzyme possesses the bifunctional property of efficiently transferring both GalNAc and Gal?1-3GalNAc residues through a linkage to the Ser or Thr.The effects of initial substrate concentration,pH,temperature,and reaction time on transglycosylation activities of BLLJ0788 were studied in detail.The enzyme was used at concentrations of 0.2 mg/mL.With the use of 5 mM GalNAc-?-pNP or Gal?1-3GalNAc-?-pNP as the donor and 200 mM Ser or 250 mM Thr as the acceptor in phosphate buffer(pH 6.0),BbhI synthesized GalNAc-?-Ser and GalNAc-?-Thr at maximal yields of 74.3%and 35.6%at 55? and 60 min,respectively.The peak values of the yield of Gal?1-3GalNAc-a-Ser and Gal?1-3GalNAc-?-Thr were 73.7%and 58.8%at 55? and 20 min,respectively.BLLJ0788 can also synthesize Gal?1-3GalNAc-STAPPA and GalNAc-STAPPA with the polypeptide as the acceptor and thus provides a powerful synthetic tool to obtain biologically important glycans.