Insight Into The Mechanisms Of Epimerization And Isomerization In Cellobiose 2-Epimerase

Cellobiose 2-epimerase,together with the N-acetyl-glucosamine 2-epimerase(AGE,EC5.1.3.8),Yih S and Mannose isomerase(MI,EC 5.3.1.7),comprised the AGE superfamily.AGE could catalyze the interconversion of the N-acetyl-glacosamine and the N-acetyl-mannosamine.MI could interconvert the mannose into fructose.And similar with MI,Yih S mainly catalyzesthe interconversion of the mannose and the fructose,with slight mount of glucose ascoproduct.MostCEsfunction mainly asepimerases,converting?1,4-linked glucose at the reducing end of oligosaccharidesto a mannose moiety via C2 epimerization.The thermostable cellobiose 2-epimerasesfrom Caldicellulosiruptor saccharolyticus(CsCE),Dictyoglomusturgidum(DtCE)and Spirochaeta thermophile(StCE),however,are bifunctional enzymes,having both epimerization and isomerization activity.Generally,it wassuggested that theCE catalyzed the epimerization reaction through the cis-enediol intermediate.But all these mechanismswere hypothesized with the crystal structure,with no experimental surpports.Most importantly,the the inherent relashion between structural characteristicsand the catalytic direction of epimerization and isomerization were actually not studied.More detailed and comprehensive studieswere required in the catalytic mechanismsofCE or AGE superfamily enzymes.In thisthesis,we obtained the apo?structure of the cellobiose 2 epimerase for Spirochaeta thermophila DSM 6578(StCE),Bacillusthermoamylovoransstrain B4166(BtCE),the structure of the CsCE with the ligand?-D-glucopyranosyl-(1?4)-D-fructose and the structure of the mutant H247F with linear and circular mannose.With the method of the loop change,mutagenesis1D and 2D NMR and QM/MM,we tried to figure out the epimerization and isomerization mechanism of AGE superfamily,and the factor influencing the direction of the catalyzation.The crystalsof the BtCE and the StCE were screened by method of hanging drop vapor diffusion,with the condition of 0.2M Trimethylamine-N-oxide,0.1M Tris-HCl p H 8.5,20%PEG4000 and 0.4 M Mg Cl₂,0.1M Tris-HCl p H 8.5,25%PEG8000,respectively.Similar with enzymesin AGE superfamily,the structuresof the BtCE and the StCE were comprised of 12?heliceswith six outer helicesand six inner helicesrunning in an opposite direction.The flexible loop of the BtCE wasmissed.All ofCE,Yih S and MI have a flexible loop at the entrance of the active site,with different shapesand residue compositions.The crystalsof the CsCE with the ligand D-glucopyranosyl-?1,4-fructose were screen in the condition of 0.4 M Mg Cl₂,Bis-HCl,p H 6.5,25%PEG4000.The ligand in the CsCE have a same configuration with the corelated D-glucopyranosyl-?1,4-glucose in the RmCE,excepting of the glucopyranose residue at nonreduced end of the disaccharide.One conformation of the glucopyranoside flipped 180°,compared with the other conformation.Specifically,the flexible loop in the CsCE moved further into the active site,with tremendouschangesin the inner molecular force.It wasresidue Glu174 in CsCE that connected with the ligand.By aligning the apo-and liganding binding enzymes,it wasfound that theCEsand Yih S that shown bifunctionstended to reshape the flexible loop during the ligand binding.The CMW wasconstructed by replacing the flexible loop of the MI with the loop of CsCE(loop148-181)and mutating the Gly375(corresponding to Trp372 in CsCE)into tryptophan.Unlike the MI,the mutant CMW shown no activity toward the monosaccharide,but shown apparent activity of epimerization to the lactose(20.06 U/mg a),like mostCEs.Thissuggested that both of the MI andCE could catalyze the epimerization and isomerization and the catalytic direction washighly possiblely caused by the conformationsof the substrate in the active site.The T_m value of the CMW wasabout 59?,20?higher than MI(39?),investigating that the residuesin the flexible loop could trendously improve the thermostability of the MI.The molecular modelization of the MI and itsmutant G375W(corresponding to CsCE?Trp372)suggested thistryptophan could decrease the binding free energy and enthalpy,facilitating the recognization of disaccharides.Asthe CMW shared the same(?/?)₆ barrel and flexible loop with CsCE,their apparent epimerization catalyzation wasnot corelated with the flexible loop but the barrel and the active site.The“two-tryptophan”in the active site may immobilize the disaccharidesin a confortation related with the epimerization.Several mutantswere obtained to study the rolesof the residuesin the flexible in the direction of epimerization and isomerization.All of the mutantsshow similar epimerization activity with the CsCE(43 U/mg).But for the isomerization activity,the E174A and G176D obtained 90%and 72%activity left and the R170AF171A,N175K,N175RG176D and G176K got lessthan 15%activity left.Specifically,the V177D and I178D got isomerization of 5.71U/mg and 7.05 U/mg,about 1.7 and 1.8 timesof that of the CsCE.All of the mutantsgot no changesin the structure and T_m value.The time curve of the I178D with the substrate of the lactose suggested the rate of the lactose,lactulose and epilactose in the equilibrium wasabout26.2:63.1:10.6,similar with CsCE(28.2:60.0:11.8).It seemsthat the long chain at the site 170and 171 of the loop made the loop more stable through salt bridge and H bond,while the long chain at the middle part of the loop(site 175,176)influence the flexibility of the loop during the ligand binding.Most importantly,the negative charged residueslike Asp could reinforce the H bond at the other side of the loop,making the loop stretch toward to the site better for ligand binding and causing the improvement in the enzymatic activity.The open form of the ligand mannose wasfirst caputured by cocrystalizing the mutant H247F with mannose.The open form of the mannose in the active site support the hypothesisthat the first step of the catalyzation of AGE family isto open the pyranose ring.The residue His377 nearby the O5 of the fructose of the ligand in CsCE wasrecommended involved in the ring open.The opening of the pyramose residueswassuggested to be conducted by the residue His377 independently and the linearization of the furanose residue wascatalyzed by the two resideusof the His377 and the His188 by investigating the H bondsbetween the residuesand the ligand.With the investigation of the LC-MS,NMR of the monosaccahridesin the catalyzation of the CsCE and Mm MI in D₂O,the catalytic mechamismsof eipmerization and isomerization inCE and MI were suggested to be cis-enediol intermediate.With the help of the QM/MM,the coopertation of the His247 and Glu250 in the proton transfer wasconfirmed,and the catalytic pocket of theCEs,MI andCE wasmodified as“three histidine-one gluctamine acid”.Both of epimerization and isomerization wascomprised of three steps-ring opening,catalyzation and ring closing.The ring opening and closed of the pyranosyl and fructosyl residue were resectively catalyzed by His377 and His377-His188.The depronaztion and protonation of C2in the epimerization wasfound conduected by the His377 and His247/Glu250.For the isomerization,the catalyzation wasconducted by the His377 and His188.On one hand,the His188 catalyzed the proton transfer between the O2 and O1 of the ligand to catalyze the interconvetion of the two conformationsof the cis-enediol intermediate(^-O-C1=C2-OH and HO-C1=C2-O^-);one the other hand,the His377 catalyzed the depronization or protonization of the C1.