Crystal structures of D-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

D-Psicose 3-epimerase(DPEase)is demonstrated to be useful in the bioproduction of D-psicose,a rare hexose sugar,from D-fructose,found plenty in nature.Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize D-fructose to yield D-psicose with a much higher conversion rate when compared with the conventionally used DTEase.In this study,the crystal structure of the C.cellulolyticum DPEase was determined.The enzyme assembles into a tetramer and each subunit shows a(β/α)8 TIM barrel fold with a Mn2+metal ion in the active site.Additional crystal structures of the enzyme in complex with substrates/products(D-psicose,D-fructose,D-tagatose and D-sorbose)were also determined.From the complex structures of C.cellulolyticum DPEase with D-psicose and D-fructose,the enzyme has much more interactions with D-psicose than D-fructose by forming more hydrogen bonds between the substrate and the active site residues.Accordingly,based on these ketohexosebound complex structures,a C3-O3 proton-exchange mechanism for the conversion between D-psicose and D-fructose is proposed here.These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

Enhancing the thermostability of D-allulose 3-epimerase from Clostridium cellulolyticum H10 via directed evolution

d-allulose,the epimer at C-3 position of d-fructose,is a low-calorie functional rare sugar,which is regarded as one of the most potential sweeteners.At present,the main production method of d-allulose is epimerization of d-fructose by d-allulose 3-epimerase(DAE).However,industrial applications of DAE are still limited by its poor thermostability.Herein,directed evolution was applied to improve the thermostability of dAE from Clostridium cellulolyticum H10(CcDAE).Two optimal mutants D281G and C289R,exhibiting 13.80-fold and 13.88-fold t_(1/2 )values as that of wild type at 65℃,respectively,were obtained.To further enhance the thermostability,the triple mutant A107P/D281G/C289R was constructed after combina-tion of mutants D281G,C289R,and previously identified thermostability-enhanced mutant A107P.The T_(m) and optimal temperature of triple mutant were increased by 14.39℃and 5℃,respectively,compared to the wild type,meanwhile,the half-life of triple mutant was 58.85-fold as that of wild type at 65℃.Furthermore,the conversion rate of triple mutant was increased from 24.76%of wild type to 27.53%using 300 g/L d-fructose as substrate at 70℃.The effectiveness of directed evolution was verified and the triple mutant with enhanced thermostability had great application value in the large-scale production of d-allulose.