Functional Characterization Of The Putrescine N-methyltransferase From Hyoscyamus Niger

Hyoscyamus niger,a medicinal plant,belongs to the Solanaceae family.The whole plant contains tropane alkaloids?TAs?,including hyoscyamine and scopolamine,which can be used as anticholinergic drugs.Due to high cost in chemical synthesis and no promising commercial prospect,the TAs is still extracted from medicine source plants.Compared with hyoscylamine,scopolamine has better efficacy and less side effects.These features make additional value to scopolamine.Meanwhile,plants have very low TAs content.Metabolic engineering is one of important means to cultivate high-yielding TAs source plants.Understanding the knowledge of molecular biology and biochemistry of TAs biosynthesis pathway is the premise of metabolic engineering.Putrescine N-methyltransferase?PMT?catalyzes the N-methylation of putrescine to form N-methylputrescine which is an important precursor for the biosynthesis of nicotine and TAs.Numerous studies have shown that PMT is the first rate-limiting enzyme in nicotine biosynthesis.However,the knowledge of PMT in TAs biosynthesis and the regulation of polyamines remains limited.In this study,according to transcriptome database?unpublished?determined by our laboratory,a gene encoding putrescine N-methyltransferase was screened then named HnPMT.The bioinformatics and expression patterns analysis of HnPMT was conducted.The enzyme activity and kinetic properties of HnPMT were identified.Finally,suppression of HnPMT expression by VIGS in H.niger had an effect on TAs and polyamine biosynthesis.The results are as follows:Cloning and bioinformatics analysis of HnPMT.Based on the sequenced H.niger transcriptomes?data not published?,HnPMT was cloned,ligated into T vector and sequenced.As demonstrated in the study,the full-length cDNA of HnPMT was 1534bp and contained a 1014 bp coding sequence encoding a 338 amino acid residue polypeptide with a molecular weight of 37 kDa and an isoelectric point of 5.78.By multiple amino acid sequence alignment represented that HnPMT was very similar to the other plant PMTs.Our phylogenetic analysis showed that the PMT family was grouped together while the SPDS family clustered to form another group.HnPMT belongs to PMT family.According to the bioinformatics analysis,it was deduced that HnPMT might catalyze the N-methylation of putrescine.Expression analysis of HnPMT.Analyzing tissue of root and leaf profile indicated that HnPMT was expressed in roots.After H.niger hairy roots had been treated with methyl jasmonate?MeJA?-containing WPM liquid medium,the expression of HnPMT then showed a significant growth with the increase of MeJA's application time.The expression of HnPMT reached the maximum at 12 h.The study suggested that HnPMT was root-specific and MeJA-responsive gene.Purification and activity assay of recombinant HnPMT.The coding sequence of HnPMT was inserted into the prokaryotic expression vector pET28a?+?,to yield the recombinant His-tagged protein in E.coli Rossetta.The HnPMT recombinant protein was obtained by optimized induction and purification conditions.By SDS-PAGE gel electrophoresis and alignment with protein Marker,HnPMT has a molecular weight of 40 kDa.Further,the HnPMT recombinant protein was purified by nickel column for the precursor feeding experiment,and the HnPMT reaction sample,the control and the standard sample were benzoylated and determined by LC-MS.Through an LC-MS spectrum analysis,as results clearly represented,the retention time for the authentic benzoylated N-methylputrescine was 11.85 min.At the same retention time,the target compound in the HnPMT reaction sample was likewise detected.But the product was not detected in the control reaction.Next,the m/z value of HnPMT reaction product was consistent with the benzoylated N-methylputrescine?311?,demonstrating that HnPMT can catalyze putrescine into N-methylputrescine.Enzyme kinetic analysis of HnPMT.At different pH?7.5-9.5?and temperature?22?-46??,we did researches about enzyme activity of HnPMT.It was easy to find that HnPMT had the highest activity at pH 9.0 and temperature of 40?.The Michaelis–Menten curves for the velocity of HnPMT were obtained under enzymatic assays conducted in the condition of pH 9.0 at 40?,and substrate putrescine concentration was range from 0.05 mM to 5 mM.HnPMT has a K_m of 74?M,a V_maxax of 0.416 nkat·mg^-1,and a K_cat of 0.0173 s^-1.Compared with the reported enzyme kinetic parameters of PMT enzyme,the Km of HnPMT was much lower indicating that HnPMT had much higher affinity to putrescine.HnPMT had much lower K_catat value demonstrating that it has low catalytic efficiency for the substrate putrescine.As enzymatic kinetics analysis showed,HnPMT had high affinity with putrescine yet very low catalytic activity,suggesting that it was a rate-limiting enzyme involved in the TAs biosynthesis in H.niger.Inhibiton of HnPMT on TAs and polyamine biosynthesis.We did VIGS experiments to halt the expression of HnPMT in H.niger.From the qPCR analysis,HnPMT expression showed a sharp fall to c.8-fold in the HnPMT-silenced plant materials over the control.By measuring the content of N-methylputrescine—the direct product given by HnPMT and the target product of TAs,both contents decreased significantly after the HnPMT was silenced.The control plants produced N-methylputrescine at concentrations of 0.21?g/g fresh weight?FW?.In stark contrast,the HnPMT-silenced plants H.niger produced markedly less N-methylputrescine at concentrations of 0.092?g/g FW.The control plants produced hyoscyamine at concentrations of 1.30?g/g dry weight?DW?.In stark contrast,the HnPMT-silenced plants H.niger produced substantially less hyoscyamine at concentrations of 0.842?g/g DW.The results showed that silencing HnPMT reduced the synthesis of N-methylputrescine and then restricted the synthesis of downstream hyoscyamine.Meanwhile,polyamine production was also analyzed.Relative to the control,the HnPMT-silencing plants showed higher levels of putrescine,spermidine and spermine production.The metabolite analysis distinctly demonstrated that silencing HnPMT not only reduced the biosynthesis of N-methylputrescine but also disrupted the production of downstream hyoscyamine,yet it simultaneously promoted the accumulation of polyamines in the H.niger plants.In summary,HnPMT was specifically expressed in roots of H.niger,which can catalyze putrescine into N-methylputrescine.HnPMT had higher affinity for putrescine but lower catalytic activity.Silencing HnPMT reduced the biosynthesis of N-methylputrescine and hyoscyamine,yet it promoted the accumulation of polyamines.Functional identification of HnPMT facilitated the understanding of TAs biosynthesis and the regulation of polyamine metabolism.