Site-directed Mutagenesis Of P-fructofuranosidase Of Bombyx Mori And Analyses On The Active Residues

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors,such as 1,4-dideoxy-1,4-imino-D-arabinitol(D-AB1),1-deoxynojirimycin(DNJ)and 1,4-dideoxy-1,4-imino-D-ribitol.D-AB1 and DNJ are strong inhibitors of ?-glucosidases(EC.3.2.1.20);however,they do not exhibit inhibitory activity against ?-fructofuranosidases(?-FFase,EC.3.2.1.26).These sugar mimic alkaloids are highly toxic to some caterpillars,such as the eri-silkworm Samia cynthia ricini(family Saturniidae)and cabbage moth Mamestra brassicae(family Noctuidae),for which mulberry trees are not the host plant in natural conditions.However,these compounds are not toxic to larvae of the silkworm Bombyx mori(family Bombycidae),which feed only on mulberry leaves and have been reared on them for thousands of years.Although ?-FFase gene has been cloned and identified from Bombyx mori(Bm Suc1),indicating that the silkworm may use ?-FFase as glycosidase to circumvent the toxic effects of such sugar mimic alkaloids,and enable itself to feed and grow well on mulberry leaves,there is little knowledge about the core enzymatic centre and key amino acid residues or motifs that support Bm SUC1 acting as a functional sucrase.Three conserved motifs,Asn-Asp-Pro-Asn-Gly(NDPNG),Arg-Asp-Pro(RDP)and Glu-Cys(EC),each containing a key acidic residue have been implicated important in substrate binding and hydrolysis for GH32 family.To investigate the activity sites and activity specificity of Bm SUC1,we replaced the three predicted catalytic sites in Bm SUC1 to Ala,respectively,by using site-directed mutagenesis and constructed four recombinant p Fast Bac Dual plasmids including a wild type of Bm SUC1 and three mutant types.Then,the recombinant proteins were expressed by a Bac-to-Bac expression system in Bm N cells and purified to detect the ?-FFase activity of enzymes.Finally,the activity characteristic of D181 A mutant,which is markedly higher than wild-type,was further qualitatively and quantitatively analyzed by HPLC-ELSD.The results in our study are mainly listed as the following.1)The cell culture medium,cell supernatant and cell insolubilized component were collected respectively for SDS-PAGE and Western blot analysis.The result showed that most of recombinant protein was retained in cell medium.The protein molecular mass is about 56 KDa,which is consistent with its theoretical molecular weight.2)Wild-type Bm SUC1 and three mutant recombinant proteins were purified by a Ni-NTA chromatographic column.Western blot analysis showed that the four recombinant proteins were obtained at higher purity.3)For detecting the activity of recombinant enzymes,the dinitrosalicylic acid(DNS)was added to the reaction mixture for measuring the glucose liberated.The results showed that Bm SUC1 displayed maximum at p H 7.0 and the temperature of 30?.D63 A and E234 A enzymes showed no any activity in the range of p H 5-9 and the temperature of 15-60?.Interestingly,the activity of D181 A was remarkable higher than Bm SUC1 and the most optimal condition for D181 A is at p H 6.0 and the temperature of 30?.4)D181A mutant had higher affinity to the substrates of both sucrose and raffinose than wild-type Bm SUC1.The HPLC analysis showed that the mixtures contained glucose,fructose,sucrose,1-kestose and 6-kestose after reactions catalyzed by both Bm SUC1 and D181 A,indicating that the two enzymes possessed hydrolytic and transfructosyl activities,furthermore,those of D181 A were markedly higher than wild-type Bm SUC1.In a word,activities of D63 A,D181A and E234 A mutants were investigated and compared with that of Bm SUC1 in this study.According to the results,we can conclude that D63,D181 and E234 are important active residues for Bm SUC1.Inside,D63 probably acts as the nucleophile and E234 as the acid/base catalyst,while D181 seems not to be directly involved in the catalytic mechanism and most probably acts as a transition-state stabilizer.Our studies give new information to understand the enzymatic structure characteristic of Bm SUC1 and help to comparatively research ?-FFases derived from other insects.