Study On Changing Coenzyme Activity And Preference Of Glucose Dehydrogenase With Site-directed Mutations Of Structural Analysis And Multiple Sequence Alignment

Glucose Dehydrogenase(GDH)is widely present oxidoreductase in many organisms.It can be used as a diagnostic enzyme for blood glucose concentration,a key enzyme for biofuel cells and biosensors.GDH is an important enzyme in the pentose phosphate metabolism pathway in organisms,which specifically catalyzes D-glucose to produce ?-D-gluconolactone,at the same time reduces coenzyme NAD(P)+to NAD(P)H.Most biocatalysed redox reactions require the participation of NAD(P)+coenzyme.The high price of coenzyme NAD(P)+greatly limits the industrial application of redox catalysis.Coenzyme regeneration is an indispensable step to achieve the biocatalysed redox reaction,and it is an effective method to solve the industrial application of oxidoreductase.Coenzyme NAD(P)+and NAD+differs structurally by the presence of one phosphate group to 2'-hydroxyl group of NADP+,but the price of NADP+is more 7 times than that of NAD+.In this study,two glucose dehydrogenases(GlcDH and GDH223)derived from B.megatherium IWG3 and 223,respectively,were used as the research objects.Through GDH structure analysis and sequence alignment of coenzyme binding site,the coenzyme specificity was efficiently switched by site-directed mutagenesis.The preferred method is to obtain mutants with high enzyme activity and preference for NADP+,which can be widely used for the regeneration of coenzyme NADPH.The main research contents of this paper are as follows:1.Based on the analysis of GlcDH structure and coenzyme binding site from B.megatherium IWG3,the 17 site of amino acid sequence of GlcDH,which bind with 2-phosphate and 3'-hydroxyl group of NAD(P)+coenzyme,is selected to perform site-specific saturation mutation.Nine mutants were screened and had significant changes of coenzyme activity and preference compared with that of wild type.The preference of mutant T17G was switched from NAD+to NADP+.Compared with the wild type GlcDH,the NADP+activity of mutant T17G increased by 15.4%,and the NAD+acitivity decreased by 25%.The coenzyme NADP+of Km of T17G increased 1.71 times than that of the wild type.The best mutant T17G exhibited an 11 fold reversal of the coenzyme preference from NAD+to NADP+.The molecule docking mutant T17G with coenzyme revealed that the replacement of Thr with Gly resulted in an increase in the binding affinity of the mutant T17G towards NADP+.The results proved that the coenzyme preference of GlcDH can be effectively switched via site-directed mutation of 17 site in GlcDH.2.Sequence alignment and structural analysis of glucose dehydrogenase family showed that 17 site of amino acids residues are located in the coenzyme binding conserved region(GXXXGXG)and play an important role in 2-phosphate interaction and 3'-hydroxyl group.We propose a strategy for determining mutation sites by multiple sequence alignments and structural analysis of coenzyme recognition regions of glucose dehydrogenase,and coenzyme activity and preference of GDH could be efficiently switched by site-directed mutations at 17 sites.The GDH233 derived from B.megatherium AS223 was used to verify the feasibility of the strategy.A site-specific mutation of Lys17 sites of GDH223 was mutated into neutral and acidic amino acids,and three single mutants GDH223 K17G,K17D,and K17T were successfully constructed.Compared with the wild enzyme,the coenzymes activity and preference of the three mutants were significantly improved.Among them,the coenzyme activity of mutant K17T increased 280.4%on NAD+and 99.2%on NADP+compared with that of the wild type.The best mutant K17D exhibited 1.94 times reversal of the coenzyme preference from NAD+to NADP+.The optimum temperature and pH for wild-type and mutants were 50? and 7.0,respectivitly.The results of site-directed mutagenesis(GlcDH and GDH223)proved that our proposed strategy can be effectively applied to switch coenzyme preference of glucose dehydrogenase family.3.To further enhance coenzyme activity and preference of K17T,site-directed mutations were performed on the adjacent 16-site of GDH223 within coenzyme binding conserved region(GXXXGXG).The experiment used mutant GDH223 K17D as a template,and the acidic glutamic and alkalin lysine were introduced to the position 16 to successfully construct double mutants S16K/K17D and S16E/K17D.The coenzyme activity of S16K/K17D improved 27.51%on NADP+.The mutant S16K/K17D exhibited 2.61 times reversal of the coenzyme preference from NAD+to NADP+.The optimum temperature and pH of the S16K/K17D were 50? and 7.0,respectively.In summary,this paper proposes the strategy of sequence alignment and structure analysis to determine the 17 site as a key site for changing coenzyme activity and preference of glucose dehydrogenase.The site-directed mutagenesis of the 17 sites of GlcDH and GDH223 could effectively switch coenzyme activity and preference according to the strategy.Without building a large number of mutant libraries and high-throughput screening,this strategy can be effectively applied to change the coenzyme preference of the glucose dehydrogenase family.