Metabolic characterization of Hyoscyamus niger root-specific putrescine N-methyltransferase

Chen, Geng; Zhao, Tengfei; Yang, Chunxian; Zhang, Qiaozhuo; Bai, Feng; Zeng, Junlan; Zhang, Fangyuan; Liu, Xiaoqiang; Lan, Xiaozhong; Chen, Min

doi:10.1016/j.plaphy.2018.03.001

Cited by 18 publications

(20 citation statements)

References 43 publications

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“…However, all three genes— HnPMT (Figure 4B), HnTRI (Figure 4C), and HnH6H (Figure 4D)—were specifically expressed in the roots of H . niger , consistent with earlier reported findings (Hashimoto and Yamada, 1987; Hashimoto et al, 1992; Geng et al, 2018). Unlike the root-specific expression of those three TA biosynthesis genes, HnODC was expressed in both roots and leaves, but its expression level was still much higher in roots than leaves (Figure 4A).…”

Section: Resultssupporting

confidence: 93%

“…The detection methods used here were similar to those applied in the enzymatic assays described below. Tropane alkaloid content was quantified by adhering to previously described methodologies (Qiang et al, 2016; Zhao et al, 2017; Geng et al, 2018). For this, 200 mg of dry powder from each plant part was accurately weighed for alkaloid extraction and detection, with at least three independent plants used for metabolite detections.…”

Section: Methodsmentioning

confidence: 99%

“…To test the catalytic activity of HnODC, its purified form was tested in a 10-mM Hepes buffer (pH 8.0) containing 1 mM dithiothreitol and 1 mM pyridoxal phosphate (PLP) for 60 min, by using different concentrations of L-ornithine as the substrate. The products (putrescine) from enzymatic assays and authentic putrescine were benzoylated into benzoyl putrescine (as carried out by Geng et al, 2018). The benzoylated samples were used for the HPLC analysis or stored at -80°C for future use.…”

Section: Methodsmentioning

confidence: 99%

“…niger as well as other plants species (Figure 1). These enzymes include putrescine N -methyltransferase (Liu et al, 2005; Kai et al, 2009b; Geng et al, 2018), tropinone reductase I (Nakajima et al, 1993b; Kai et al, 2009a; Qiang et al, 2016), tropinone reductase II (Hashimoto et al, 1992; Nakajima et al, 1993a,b), CYP80F1 (Li et al, 2006), and hyoscyamine 6β-hydroxylase (Matsuda et al, 1991; Li et al, 2012). Since their identification, the associated TA-biosynthesis genes have been applied to engineer TA biosynthesis in plants via the overexpression method (Zhang et al, 2004; Wang et al, 2011; Zhao et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Metabolic Characterization of Hyoscyamus niger Ornithine Decarboxylase

Zhao¹,

Wang²,

Bai³

et al. 2019

Front. Plant Sci.

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Ornithine decarboxylase (ODC) catalyzes ornithine decarboxylation to yield putrescine, a key precursor of polyamines, and tropane alkaloids (TAs). Here, to investigate in depth the role of ODC in polyamine/TA biosynthesis and to provide a candidate gene for engineering polyamine/TA production, the ODC gene ( HnODC ) was characterized from Hyoscyamus niger , a TA-producing plant. Our phylogenetic analysis revealed that HnODC was clustered with ODC enzymes of plants. Experimental work showed HnODC highly expressed in H. niger roots and induced by methyl jasmonate (MeJA). In the MeJA treatment, the production of both putrescine and N -methylputrescine were markedly promoted in roots, while contents of putrescine, spermidine, and spermine were all significantly increased in leaves. By contrast, MeJA did not significantly change the production of either hyoscyamine or scopolamine in H. niger plants. Building on these results, the 50-kDa His-tagged HnODC proteins were purified for enzymatic assays. When ornithine was fed to HnODC, the putrescine product was detected by HPLC, indicating HnODC catalyzed ornithine to form putrescine. Finally, we also investigated the enzymatic kinetics of HnODC. Its K m , V max , and K cat values for ornithine were respectively 2.62 ± 0.11 mM, 1.87 ± 0.023 nmol min -1 μg -1 and 1.57 ± 0.015 s -1 , at pH 8.0 and at 30°C. The HnODC enzyme displays a much higher catalytic efficiency than most reported plant ODCs, suggesting it may be an ideal candidate gene for engineering polyamine/TA biosynthesis.

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Section: Resultssupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Metabolic Characterization of Hyoscyamus niger Ornithine Decarboxylase

Zhao¹,

Wang²,

Bai³

et al. 2019

Front. Plant Sci.

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“…Accordingly, there is a longstanding interest in elucidating the TA biosynthetic pathway. Most enzyme‐catalysed reactions associated with TA biosynthesis have been characterized, including those related to putrescine N ‐methyltransferase (PMT), N ‐methylputrescine oxidase (MPO), type III polyketide synthase (PYKS), tropinone synthase (CYP82M3), tropine‐forming reductase (TRI), aromatic amino acid aminotransferase (AT4), phenylpyruvic acid reductase (PPAR), littorine mutase (CYP80F1), and hyoscyamine 6β‐hydroxylase (H6H) (Li et al , ; Katoh et al , ; Bedewitz et al , ; Qiang et al , ; Geng et al , ; Qiu et al , ) (Fig. ).…”

Section: Introductionmentioning

confidence: 99%

Functional genomics analysis reveals two novel genes required for littorine biosynthesis

et al. 2019

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Some medicinal plants of the Solanaceae produce pharmaceutical tropane alkaloids (TAs), such as hyoscyamine and scopolamine. Littorine is a key biosynthetic intermediate in the hyoscyamine and scopolamine biosynthetic pathways. However, the mechanism underlying littorine formation from the precursors phenyllactate and tropine is not completely understood.Here, we report the elucidation of littorine biosynthesis through a functional genomics approach and functional identification of two novel biosynthesis genes that encode phenyllactate UDP-glycosyltransferase (UGT1) and littorine synthase (LS).UGT1 and LS are highly and specifically expressed in Atropa belladonna secondary roots. Suppression of either UGT1 or LS disrupted the biosynthesis of littorine and its TA derivatives (hyoscyamine and scopolamine). Purified His-tagged UGT1 catalysed phenyllactate glycosylation to form phenyllactylglucose. UGT1 and LS co-expression in tobacco leaves led to littorine synthesis if tropine and phenyllactate were added.This identification of UGT1 and LS provides the missing link in littorine biosynthesis. The results pave the way for producing hyoscyamine and scopolamine for medical use by metabolic engineering or synthetic biology.

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