Metabolic characterization of Hyoscyamus niger root-specific putrescine N-methyltransferase
Metabolic characterization of Hyoscyamus niger root-specific putrescine N-methyltransferase
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黑天仙根根特异性腐胺 N-甲基转移酶的代谢特征
DOI：
10.1016/j.plaphy.2018.03.001
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发表时间：
2018-06-01
影响因子：
6.5
通讯作者：
Liao, Zhihua
中科院分区：
文献类型：
--
作者：
Geng, Chen;Zhao, Tengfei;Liao, Zhihua
N-甲基腐胺是尼古丁和药物托烷生物碱的前体，如山梨碱。腐胺N-甲基转移酶(PMT)催化腐胺N-甲基化生成N-甲基腐胺。虽然PMT在尼古丁生物合成中的作用是明确的，但对PMT在托烷生物碱(TM)生物合成和多胺调节中的了解仍然有限。我们鉴定了一个来自黑夜蛾的PMT基因，命名为HnPMT，它在根中特异表达，特别是在次生根中表达，并受到茉莉酸甲酯(MeJA)的显著诱导。当GUS基因由HnPMT的5‘侧翼启动子区域驱动时，GUS基因在拟南芥的根或黑曲霉毛状根培养的维管组织(包括中柱和内皮层)中特异表达。对重组HnPMT进行纯化，用于酶活性检测。经LC-MS确证，HnPMT将腐胺转化为N-甲基腐胺。动力学分析表明，HnPMT与腐胺有较高的亲和力，但催化活性较低，是一种限速酶。当用VIGS方法抑制黑曲霉HnPMT时，N-甲基腐胺和山梨碱的含量显著降低，而腐胺、亚精胺和精胺和温敏胺混合物的含量显著增加，这表明HnPMT参与了托烷生物碱的生物合成，并在调节多胺的生物合成中起到了很好的作用。HnPMT的功能鉴定有助于理解TA的生物合成，从而暗示HnPMT催化的步骤可能是黑曲霉TA合成代谢工程的靶点。
N-methylputrescine is the precursor of nicotine and pharmaceutical tropane alkaloids such as hyoscyamine. Putrescine N-methyltransferase (PMT) catalyzes the N-methylation of putrescine to form N-methylputrescine. While the role of PMT in nicotine biosynthesis is clear, knowledge of PMT in the biosynthesis of tropane alkaloids (TM) and the regulation of polyamines remains limited. We characterized a PMT gene from Hyoscyamus niger, designated HnPMT that was specifically expressed in roots, especially in the secondary roots and dramatically induced by methyl jasmonate (MeJA). The GUS gene was specifically expressed in Arabidopsis roots or in the vascular tissues, including pericycles and endodermis, of the H. niger hairy root cultures, when it was driven by the 5'-flanking promoter region of HnPMT. The recombinant HnPMT was purified for enzymatic assays. HnPMT converted putrescine to form N-methylputrescine, as confirmed by LC-MS. The kinetics analysis revealed that HnPMT had high affinity with putrescine but low catalytic activity, suggesting that it was a rate-limiting enzyme. When HnPMT was suppressed in the H. niger plants by using the VIGS approach, the contents of N-methylputrescine and hyoscyamine were markedly decreased, but the contents of putrescine, spermidine and a mixture of spermine and thermospermine were significantly increased; this suggested that HnPMT was involved in the biosynthesis of tropane alkaloids and played a competent role in regulating the biosynthesis of polyamines. Functional identification of HnPMT facilitated the understanding of TA biosynthesis and thus implied that the HnPMT-catalyzed step might be a target for metabolic engineering of the TA production in H. niger.