Cloning And Functional Analysis Of Two Distinct Tropinone Reductases In Przewalskia Tangutica

Tropane alkaloids?TAs?are widely used anticholinergic drugs,which are secondary metabolites mainly found in solanaceae species.TAs used in the markets mainly depend on the extraction of the plants,and which are low production and expensive.Therefore,it is meaningful to use metabolic engineering method to increase the production of TAs in the plants.What's more the development of genetic engineering makes it possible to transform TAs biosynthetic pathway by means of metabolic engineering,and the application of metabolic engineering depends on the biological chemical and molecular biological studies on the metabolic engineering of the TAs.Przewalskia tangutica is a traditional Chinese medicinal plant which is mainly planted in the Qinghai-Tibet plateau.Its root contains rich hyoscyamine?main alkaloid?.The content of hyoscyamine is 1.67%to 3.82%of dry weight and the total alkaloid content is 2.06%to 4.01%of dry weight.P.tangutica is one of the ideal TAs medicinal source plants.So far,there have been few reports on the genes for biosynthesis pathway of TAs in P.tangutica.Tropinone reductases?TRs?are divided into tropinone reductase I?TRI?and tropinone reductase II?TRII?.TRI and TRII as a branch point in the biosynthesis pathway of tropane alkaloids which can reduce tropinone to tropine and pseudotropine respectively.The tropine is a direct source of tropine rings in the molecular structure of the tropane alkaloids.However,pseudotropine,as a branch pathway,let tropane flow to other secondary metabolites.In this study,P.tangutica was used as research subject to analyze and compare other reported tropinone reductase gene sequences of Solanaceae plants.By using the technology of the RACE,the full length cDNA of PtTRI is 1176bp,including 822 bp coding region and 70 bp 5?UTR,284 bp 3?UTR and termination codon is TGA.The full length cDNA of PtTRII is 981 bp,including 783 bp coding region and 87 bp 5?UTR,111 bp 3?UTR and the termination codon is TAA.The products of PtTRI and PtTRII catalyzed reaction were identified by gas chromatography mass spectrometry?GC-MS?analysis,and the results showed that the PtTRI and PtTRII have biological functions and let tropinone be reduced to the tropine and pseudotropine respectively.Present study comprises the construction of PtTRs protein expression vectors and E.coli Rosetta overexpression of two tropinone reductase genes from P.tangutica.The enzyme kinetic parameters of the recombinant proteins were determined.The optimum pH points of the reduction reaction and oxidation reaction of recombinant PtTRI protein were 6.4 and 10.6 respectively.The optimum pH of the reduction reaction of recombinant PtTRII protein was 6.8.According to the enzymatic kinetic assaying and the Michaelis-Menten equation,the Km and Kcat/Km of PtTRI were respectively 0.52±0.10 mM,42.26 s^-1·mM^-11 at pH 6.4?physiological activity of plants?when tropinone was used as substrate.Moreover,the Km and Kcat/Km of PtTRI for tropine were 0.07±0.00 Mm,100.11 s^-1·mM^-1 at pH 10.6 respectively.The results show that PtTRI has a higher affinity and catalytic efficiency when substrate was tropine,however the enzymatic kinetic assaying for tropine was done in pH 10.6,a strongly alkaline environment,while plants cells are in a weakly acidic environment,so in vivo of the P.tangutica,the reduction is mainly accounted for dominance.The Km,Kcat/Km values of PtTRII for tropinone were 0.087±0.02 mM,116.90 s^-1·mM^-1,respectively,indicating that PtTRII has a higher affinity and reductive activity for tropinone than PtTRI.Gene expression of profiling by qPCR revealed that the PtTRI transcript was expressed in all three vegetative organs of P.tangutica with highest expression level in roots and relatively lower abundance on stems and leaves which has similar expression level with the other TAs plants related genes PMT and H6H,while PtTRII had higher expression level in roots and stems.The PtTRI expression level suggests that the biosynthesis of TAs occurred not only in the roots but may also in the aerial parts of P.tangutica.In this study,we identified the characterization of PtTRI and PtTRII,and compared TRI activities of P.tangutica and other TAs species like Anisodus luridus,Brugmansia arborea and Datura stramonium.From the results,we can may determine that tropinone reductase I of P.tangutica is a more efficient enzyme,and this result can provide new ideas for regulation of synthesis and metabolism of TAs.