Functional Analysis Of Tropinone Reductase Like Genes In Atropa Belladonna

Hyoscyamine and scopolamine belong to tropane alkaloids (TAs), which are usually extracted from the plants of Solanaceae family, such as Atropa belladonna. Tropane alkaloids are widely used as anti-cholinergic drugs. Compared to hyoscyamine, Scopolamine has fewer side effects and stronger bioactivities, which is urgently demanded in the word market. Atropa belladonna is the main medicinal herbal resource of TAs for commercial values. However, A. belladonna has very low content of hyoscyamine and scopolamine. So, to develop medicinal herb resource plants with high-yield tropane alkaloids is always our aims. In the past decades, the researchers tried to develop A. belladonna with higher levels of tropane alkaloids through the methods of virus free and crossing, but failed. With the development of metabolic engineering, it is possible to genetically modify the biosynthetic pathway at the molecular levels, and metabolic engineering is absolutely dependant on the discoveries of molecular biology and biochemistry. Based on the previous results of EST sequencing, we cloned the cDNA of a tropinone reductase like gene by RACE from Atropa belladonna, named AbTRL1, and the bioinformatics analysis was carried out. In previous experiment, we also cloned a full-length cDNA of another TRL named AbTRL2 which might be tropinone reductase Ⅱ according to bioinformatics analysis, but it was not been functionally identified. In the present study, we constructed the digital expression profiling and inducing profiling of the two genes, and then their recombinant proteins were respectively purified from Escherichia coli and used for enzymatic assays; further, site-specific mutagenesis on the specific site of AbTRL1 was performed and enzymatic assay were investigated. The final result indicated that AbTRL2 had catalytic activity both on tropinone and pseudotropine. AbTRLl had no catalytic activity both on tropinone and pseudotropine, but certain catalytic activity was detected when site-specific mutagenesis was performed.Tropinone reductase is the key enzyme on the branching point of biosynthetic pathway of TAs, containing TRⅠ and TRⅡ. The 3-carbonyl of tropinone can be reduced to α-hydroxy to form tropine by TRⅠ, but the 3-carbonyl of tropinone can be reduced to β-hydroxy to form ψ-tropine by TRⅡ. We cloned the full-length cDNA of AbTRL1 from Atropa belladonna. The full-length cDNA of AbTRL1 was 1078 bp, containing an ORF of 816 bp,5’UTR of 14 bp and 3’UTR of 248 bp and a polyA of 15 bp. The coding sequence of AbTRL1 encoded a 271-amino-acid polypeptide with the calculated molecular weight of 29.7 kD and the pI of 6.75.Bioinformatics analysis of AbTRLl showed that it had 80% similarity with the reported tropinone reductases of Solanaceae. Phylogenetic analysis indicated that its genetic distance was between TR I and TRⅡ s, and especially closer to TR I s. The conserved domain prediction showed that there was a short-chain dehydrogenase domain, suggesting that it belonged to SDR family just like the TRs in mandala and belladonna. Spacial structure prediction showed that there were conserved amino acid residues of Lys29�'Lys31 and Arg51�'Arg53 at the N-terminal for SDR binding to NADPH. The amino acid triad, Ala1、His112 and Val168 at the substrate tropinone binding region, which were important for substrate binding to the enzyme, was consistent to belladonna only at Ala158�'Ala160, but the Tyr110�'His112, Leu166�'Val168 were not consistent. Digital expression profiling and inducing profiling indicated that AbTRL1 had expression in different organs with no significant difference, but lower expressing levels in leaves. So it might be a constitutive expression gene. Finally, both of AbTRLl and AbTRL2 showed reponse to ABA treatment.The prokaryotic expression vectors were constructed to identify the function of AbTRL1 and AbTRL2 gene. The soluble protein were successfully induced by 1 mmolL1 IPTG for 6 h. The recombinant AbTRLl and AbTRL2 were respectively about 30 kD and 28 kD. Recombinant protein AbTRLl and AbTRL2 were purified, and then their reduction reaction activity were detected using tropinone,4-methylcyclohexanone and 3-quinuclidinone hydrochloride as substrate in the presence of NADPH. Besides, their oxidation reaction activity was also detected using tropine and pseudotropine as substrate in the presence of NADP+. The enzymatic assay showed that AbTRLl protein could catalyze the 4-methylcyclohexanone, but not tropinone, tropine and 3-quinuclidinone hydrochloride. Nevertheless, the AbTRL2 protein had catalytic activity on tropinone,4-methylcyclohexanone and pseudotropine.The specific site (Tyr at 110 positon changed into His) of AbTRLl was site-specific mutated to construct prokaryotic expression vector named as pET28-AbTRL1 Y110H. Then, the prokaryotic expression vector was obtained by Rosetta to get the soluble protein AbTRL1 Y110H by IPTG induction. The enzymatic assay showed that AbTRL1 Y110H had catalytic activity to catalyzed tropinone to form tropine.Functional analysis of AbTRL1 and AbTRL2 in the present study indicated that AbTRL2 gene encoded tropinone reductase Ⅱ (namely pseudotropine forming reductase). Besides, AbTRL1, a tropinone reductase like gene, was firstly founded and suggested that there was a close relationship with others tropinone reductase in evolutionary history through the analysis of the bioinformatics. The original AbTRL1 did not have the enzymatic activity to catalyzed tropinone, but its mutant (Y110H) had the enzymatic activity to catalyze tropine and this suggestd that His110 was an essential amino acid for tropinone reductase. In summary, this research provided an experimental basis for further understanding of TRs.