Mining Of New Lignan-related UDP-glycosyltransferses In Isatis Indigotica And Enzymatic Study In Vitro

Isatis indigotica Fort.,belonging to the family Cruciferae,is a conventional Chinese herbal medicine.The root of I.indigotica is traditional Chinese medicine,named as ’Ban-Lan-Gen’,whose clinical indications is influenza.Modern pharmacological studies show that isatis root has anti-inflammatory and antiviral effects.As one of the main bioactive components in isatis root,lignans and glycosides have strong antiviral effects,among which different forms of glycosylation can also lead to differences in antiviral activities.Glycosyltransferases in plants are catalyzed by Uridine diphosphate glycosyltransferases(UGT),However,only IiUGT71B5a and IiUGT71B5b have been reported so far.In previous study,we identified three ligninrelated UGTs(including IiUGT71B5a)from I.indigotica,IiUGT71B5a from I.indigotica was found to catalyze the formation of mono-glycoside or di-glycoside of pinoresinol from I.indigotica,but its activity to lariciresinol was relatively low.It indicates that there may be other lignan glycosyltransferases in I.indigotica,which needs to be systematically explored and verified.Based on the transcriptome and genomic data of I.indigotica,we cloned the predicted UGT genes.By enzymatic screening with lignans as the substrate,we obtained a new lignan glycosyltransferase,temporarily named UGT349.The phylogenetic tree showed that UGT349 belongs to UGT90 family,which was far from the previously reported plant lignan glycosyltransferase,and may represent a new kind of lignan glycosyltransferase.In order to study its enzymatic properties and biological functions,recombinant plasmids carrying different tags were constructed for protein expression and purification.By transiently overexpressing UGT349 was tryied in tobacco leaves.UGT349 overexpression and RNAi vectors were constructed to further explore the function and location in plants.Phloridzin,as a kind of flavonoids,mainly exists in apple,sweet tea and other plants.It is produced by the substrate phloretin catalyzed by P2’GT glycosyltransferase.It has antibacterial,antiviral,whitening and other bioactivities.At present,there are few reported plant P2’GT.This study found that our previously identified lignan glycosyltransferase UGT236(IiUGT71b5a)can catalyze the efficient conversion of phloretin to phloridzin in vitro.Through the prokaryotic expression system,the recombinant protein was obtained by inducing the expression and purification for its enzymatic studies.The key active sites of UGT236 were revealed by homology modeling,molecular docking and site directed mutation.Based on the systematic screening,a new lignan glycosyltransferase UGT349 was obtained,which broadened the types of lignan glycosyltransferase in I.indigotica.In addition,we found that a previously cloned lignan glycosyltransferase UGT236(IiUGT71b5a)can catalyze phloretin to produce phloridzin,which not only reflects the substrate promiscuity of glycosyltransferase,but also provides a efficient gene module of phloridzin glycosyltransferase for synthetic biology.Through homology modeling,molecular docking and site directed mutation,it reveals the selective formation of phloridzin and trilobatin,which provides a theoretical basis for the subsequent directional design to obtain high active and specific UGT.The main results of this study are as follows:1.Identification of new lignan glycosyltransferase from I.indigotica(1)A new lignan glycosyltransferase UGT349 was obtained through systematic and activity screening from I.indigotica.The amino acid sequence homology was compared with the reported lignan UGT,and the phylogenetic tree was constructed.It was found that UGT349 belonged to UGT90 family,which is far from the reported plant lignan glycosyltransferase.The ORF region of UGT349 gene was amplified and the prokaryotic expression vector was constructed.The recombinant protein was induced to express and purified from E.coli for in vitro enzymatic assay.UGT349 could glycosylate lariciresinol to generate lariciresinol-4’-O-β-D-glycoside and lariciresinol-4-O-β-D-glycoside.(2)Bioinformatics analysis showed that the ORF region of the cloned UGT349 gene was 1 401 bp,encoding 467 amino acids.The predicted isoelectric point(PI)of the protein was 5.96,and the relative molecular weight(MW)was 52.77 kDa.It was a stable protein with strong hydrophobicity,and it might be a non-secreted protein or a non-membrane protein.The secondary structure mainly includes a helix,β sheet and irregular curls.(3)Real-time PCR analysis showed that UGT349 was expressed in I.indigotica root,stem,leaf and tender leaf,and the expression level in stem was the highest,which was about twice as high as that in root.(4)In order to further explore the biochemical characteristics of UGT349 and purify the UGT349 recombinant protein,His label was first used to try to purify the UGT349 recombinant protein with phosphate buffer system.It was found that the amount of recombinant protein purification was low,and the purification system was replaced with Tris-HCl buffer system,but still can’t get a lot of recombinant proteins.MBP tag and GST tag were also used for purification,but the amount of recombinant protein was still too small and the recombinant protein purified by MBP tag had catalytic activity.Finally,we tried to add His tag at the N-terminal,express and purify the recombinant protein,and the amount of recombinant protein obtained increased,but it has no catalytic activity.(5)The subcellular localization showed that UGT349 protein was localized in cell membrane and nucleus.(6)In order to further study the function of UGT349 in I.indigotica,the overexpression and interference vector of UGT349 were constructed to induce the transgenic hairy roots of I.indigotica,laying a foundation for further elucidate the function of UGT349 in the biosynthesis pathway of lignans.2.Enzymatic study on catalyzing phloridzin by lignan glycosyltransferase UGT236 from I.indigotica(1)In vitro assay showed that UGT236 was also able to transform phloretin into phloridzin.Phylogenetic analysis showed that UGT236 had the highest homology with P2’GT glycosyltransferase UGT71A15.These results suggested that UGT236 may be a potential P2’GT.(2)In order to further explore the catalytic properties of UGT236,a prokaryotic expression vector of UGT236 was constructed to induce the expression and purification of recombinant protein.The enzymatic properties of UGT236 were investigated by using the purified recombinant protein.The results showed that the optimal reaction time of UGT236 was 12 h,and the relative conversion rate was about 80%.The optimal reaction temperature is 40℃ and the relative conversion is 20%.The best reaction system was Na2HPO4-NaH2PO4(pH=8.0),and the relative conversion rate was about 30%.The results showed that Fe2+,Mn2+and Co2+could improve the catalytic activity of UGT236,Al3+and Ni2+could partially inhibit the catalytic activity of UGT236,but Mg2+,Na+,Ca2+,Li2+and K+had little effect.Enzyme kinetic parameter experiments showed that UGT236 had lower affinity and catalytic efficiency for phloretin than MdP2’GT,UGT88F1 and UGT71A15,but its product was single and could be used as one of the catalytic elements for phloridzin synthesis in vitro.(3)Homology modeling and molecular docking were used to screen some possible active sites.Site-directed mutation showed that E391A,H15A and T141A mutants lost the catalytic activity,while the activity of S142A and triple mutant LAE_QTD(L388Q/A390T/E391D)almost disappeared.The conversion rates of UGT236 was reduced after mutation at other sites.Interestingly,F146A mutation produced trilobatin.Saturated mutations of Phe146 site showed that the activity of F146C and F146R was completely lost.Similar to wild type,the mutant F146K produced only product phloridzin.Molecular docking revealed that His 15 fixed the first phenyl in phloretin through Π-Π stacking,Glu391 or Thr141 bonded to the 6’OH in the second phenyl in phloretin through Π-Π stacking,and Phe146 reduced the distance between 2’OH and UDP-glucose through Π-Π stacking.This observation facilitated 2’-OH to be substituted by a sugar to form the product phloridzin.Therefore,the single mutants H15A,E391A and T141A were inactivated due to the loss of fixation of phloretin.The inactivation of F146C and F146R may be due to enzyme instability caused by residue oxidation and steric hindrance caused by excessive residue on substrates,respectively.At 146 site of UGT236,Lys has a great agreement with the Π-Πstacking effect of Phe on the second phloretin benzene ring,so F146K exhibited similar activity with WT.Compared to Phe,both Ala and Asn were too small to act on the second benzene ring of phloretin,resulting in almost the same production of phloridzin and trilobatin.In other 146 site mutants,the interaction force between 146 and the second benzene ring of phloretin is less than the Π-Π stacking and cation-Πinteractions of residues Phe and Lys,resulting in the formation of trilobatin in the product,but the content of trilobatin is lower than that of phloridzin.In conclusion,this study identified a new lignan glycosyltransferase UGT349 from I.indigotica by systematic screening and explored its partial catalytic characteristics,providing a research basis for the subsequent interpretation and exploration of its functions in vivo.In addition,this study broadened the knowledge of lignan glycosyltransferase UGT236 of I.indigotica and determined its enzymatic properties to substrate phloretin.Homology modeling and molecular docking and site-directed mutagenisis provided possible explanations of the structural basis of product diversity.These results provided reference for the subsequent directed design of phloretin-2’-O-glycotransferase and phloretin-4’-O-glycotransferase.