Study On Flavonoid Prenyltransferases From Glycyrrhiza Uralensis

Licorice, derived from the dried roots and rhizomes of Glycyrrhiza speices (Leguminosae family), such as Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L., is one the most frequently used herbal medicine in TCM. It is mainly used to treat peptic ulcer, cough, hepatitis C and intoxication. More importantly, it is used to moderate and harmonize the characteristics of other herbs in TCM. To date, more than200prenylated flavonoids have been identified from licorice, and many of them exhibited a variety of bioactivities. Prenyltransferase, as the key enzyme involving in their biosynthesis, catalyzes the prenylation of the flavonoid skeletons via a Friedel-Crafts alkylation. However, no flavonoid prenyltransferase has been identified in Glycyrrhiza sp.Membrane-bound plant flavonoid prenyltransferases possess strict substrate specificity, which bring difficulties in gene cloning, hererogenous expression and functional characterization. Divergent from the structurally diverse prenylflavonoids, only six flavonoid prenyltransferases have been identified. It is expected that more prenyltransferases will be identified and the biosynthetic pathways of natural prenylflavonoid need to be clarified. In this thesis, prenyltransferases from G. uralensis have been systematically studied and the following results have been obtained:1. Prenylation of flavonoids by cultured cells and their microsome of G uralensisThe cultured cells of G. uralensis conducted the prenylation of chrysin and apigenin, the microsomal fraction presented the prenylation of diomestin,2’-hydroxygerinein, isoliquiritigenin,4-hydroxychalcone,4’-hydroxychalcone, phloretin. Elicitor methyl jasmonate up-regulated the expression of prenyltransferase due to the increasement of conversion rate of prenylated apigenin. These results indicated that there existed plant flavonoid prenyltransferases in the cultured cells of G. uralensis. And it was also predicted that various kinds of flavonoid prenyltransferases or prenyltransferases with substrate promiscuity were present and responsible for the prenylation of various flavonoids.2. Cloning and characterization of flavonoid prenyltransferase genes from G uralensisThree degenerate oligonucleotide primer pairs were designed according to the sequence information of known plant flavonoid prenyltransferases to isolated candidate genes from G. uralensis.8novel cDNA sequences were obtained by RT-PCR coupled with RACE (Rapid Amplification cDNA Ends).The deduced polypeptides were analyzed by bioinformatics and the results showed that:1) The candidate polypeptides possessed two aspartate-rich motifs that are conserved in prenyltransferases, NQXXDXXXD and KD(I/L)XDX(E/D)GD;2) The polypeptides possessed a putative transit peptide sequence for targeting the plastids;3) They were predicted as9trans-membrane proteins;4) They were grouped into the plant flavonoid prenyltransferases except for GuPT5. All deduced prenyltransferases were heterologously expressed in Saccharomyces cerevisiae and4of them have been functionally characterized:GuA6DT was responsible for the6-prenylation of flavones, GuILDT prenylated chalcones, GuPT2A-7acted on2’-hydroxygenistein, and GuPT5catalyzed2,3-dihydroxynaphthalene.3. GuA6DT is a novel prenyltransferase from G uralensis that catalyzed the regiospecific6-prenylation of flavonesG. uralensis cell suspension cultures are able to prenylate exogenously fed apigenin. And a novel prenyltransferase, responsible for this prenylation of apigenin at C-6position, was denoted GuA6DT (G. uralensis apigenin6-dimethylallyltransferase). Various flavonoids were investigated as substrates to shed light on the "enzyme-substrate structure selectivity" of GuA6DT. The results showed that:1) GuA6DT possesses strict substrate specificity. Only flavones can be recognized by GuA6DT;2) GuA6DT possesses strict regiospecificity. The prenylation occurs at C-6position selectively;3) 5-OH and7-OH are necessary in the prenylation;4) Substituents at C-3or C-8hinders the catalyzing;4) Substituents on the B-ring exert an inessential effect on prenylation. The comparison of the GuA6DT gene expression in different organs revealed that the mRNA is mainly expressed in the aerial parts. Coincidently, the prenylated apigenin was exclusively detected in the stem and leave, but not detected in the root. Moreover, the analysis of the activity of chimeric enzymes between GuA6DT and SfFPT implies that the determinant region for the observed flavones specificity might be the domain neighboring the fifth transmembrane a-helix of prenyltrnsferase.4. Prenylated chalcone is a crucial intermediate of structurally diverse prenylflavonoidsGuILDT (G_. uralensis isoliquiritigenin dimethylallyltransferase), another novel flavonoid prenyltransferase with substrate specificity, is responsible for the C-3’ prenylation of chalcone. Hydroxyls on the ring A or A’of chalcone exert an inessential effect on the prenylation.Chalcone was acted as the common intermediate of flavonoids including flavanones, flavones and isoflavones with the reason that they can be further biosynthesized by CHI (chalcone isomerase), FS (flavones synthase) and IFS (isoflavone synthase). It was hypothetized that prenylchalcone plays an important role in the biosynthesis of prenylflavonoid. In order to confirm this hypothesis, a chalcone isomerase GuCHI from G. uralensis was cloned and characterized. The results revealed that GuCHI catalyzed the cyclization to form (-)-flavanones stereo-selectively and the prenyl group did not affect the cyclization. A useful hint was given that structurally diverse prenylflavonoid may originate from the enzymatic reaction of flavonoid skeleton by tailing prenylation or from a common prenylated intermediate, such as prenylchalcone, followed by CHI, FS or IFS etal. It is first time to clarify the fact that the prenylflavanone originates from prenylchalcone. And it will pave the way to clarify the biosynthesis pathway of natural prenylflavonoids in G uralensis or even other plant. To sum up,8prenyltransferase genes were cloned following by heterologous expression in Saccharomyces cerevisiae in our studies. And4of them have been characterized, especially, GuA6DT was responsible for the6-prenylation of flavones, GuILDT prenylated chalcones at C-3’. Furthermore, it was found that a novel chalcone isomerase GuCHI, catalyzed chalcones to flavanones, and was not affected by the prenyl subsitutent on substrates. These results imply that prenylchalcone is the intermediate of prenylflavanone even other prenylated flavonoid. These studies not only paved the way for the structure and function investigation of flavonoid prenyltransferase, but also provided useful hints for the study on biosynthesis of natural prenylflavonoids. Furthermore, it would provide a new approach to enzymatic synthesis of diverse active prenylated flavonoids.