Jingqiong Wan1, Yangzhen Liao1, Jia Liu2, Wenmin Du3, Chang Liu4, Yuan Wei3, Zhen Ouyang5,6. 1. School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China. 2. College of Food Science and Technology, Yunnan Agricultural University, Kunming, 650201, China. 3. School of Pharmacy, Jiangsu University, Zhenjiang, 212013, China. 4. Nanjing Institute for Comprehensive Utilization of Wild Plants, Nanjing, 210042, China. 5. School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China. zhenouyang@ujs.edu.cn. 6. School of Pharmacy, Jiangsu University, Zhenjiang, 212013, China. zhenouyang@ujs.edu.cn.
Abstract
MAIN CONCLUSION: The novel C-methyltransferase, MaMT1, could catalyze the conversion of piperidine to 2-methylpiperidine, which may be involved in the methylation step of DNJ biosynthesis in mulberry leaves. Mulberry (Morus alba L.) is a worldwide crop with medicinal, feeding and nutritional value, and 1-deoxynojirimycin ((2R, 3R, 4R, 5S)-2-hydroxymethyl-3, 4, 5-trihydroxypiperidine, DNJ) alkaloid, a potent α-glucosidase inhibitor, is its main active ingredient. Our previous researches clarified the biosynthetic pathway of DNJ from lysine to Δ1-piperideine, but its downstream pathway is unclear. Herein, eight differential methyltransferases (MTs) genes were screened from transcriptome profiles of mulberry leaves with significant differences in DNJ content (P < 0.01). Subsequently, MaMT1 (OM140666) and MaMT2 (OM140667) were hypothesized as candidate genes related to DNJ biosynthesis by correlation analysis of genes expression levels and DNJ content of mulberry leaves at different dates. Functional characterization of MaMT1 and MaMT2 were performed by cloning, prokaryotic expression and enzymatic reaction in vitro, and it showed that MaMT1 protein could catalyze the conversion of piperidine to 2-methylpiperidine. Moreover, molecular docking confirmed the interaction of MaMT1 protein with piperidine and S-adenosyl-L-methionine (SAM), indicating that MaMT1 had C-methyltransferase activity, while MaMT2 did not. The above results suggested that MaMT1 may be involved in the methylation step of DNJ alkaloid biosynthesis in mulberry leaves, which is a breakthrough in the analysis of DNJ alkaloid biosynthetic pathway. It is worth mentioning that the novel MaMT1, annotated as serine hydroxymethyltransferase, could rely on SAM to perform C-methyltransferase function. Therefore, our findings contribute new insights into the research of DNJ alkaloid biosynthesis and C-methyltransferase family.
MAIN CONCLUSION: The novel C-methyltransferase, MaMT1, could catalyze the conversion of piperidine to 2-methylpiperidine, which may be involved in the methylation step of DNJ biosynthesis in mulberry leaves. Mulberry (Morus alba L.) is a worldwide crop with medicinal, feeding and nutritional value, and 1-deoxynojirimycin ((2R, 3R, 4R, 5S)-2-hydroxymethyl-3, 4, 5-trihydroxypiperidine, DNJ) alkaloid, a potent α-glucosidase inhibitor, is its main active ingredient. Our previous researches clarified the biosynthetic pathway of DNJ from lysine to Δ1-piperideine, but its downstream pathway is unclear. Herein, eight differential methyltransferases (MTs) genes were screened from transcriptome profiles of mulberry leaves with significant differences in DNJ content (P < 0.01). Subsequently, MaMT1 (OM140666) and MaMT2 (OM140667) were hypothesized as candidate genes related to DNJ biosynthesis by correlation analysis of genes expression levels and DNJ content of mulberry leaves at different dates. Functional characterization of MaMT1 and MaMT2 were performed by cloning, prokaryotic expression and enzymatic reaction in vitro, and it showed that MaMT1 protein could catalyze the conversion of piperidine to 2-methylpiperidine. Moreover, molecular docking confirmed the interaction of MaMT1 protein with piperidine and S-adenosyl-L-methionine (SAM), indicating that MaMT1 had C-methyltransferase activity, while MaMT2 did not. The above results suggested that MaMT1 may be involved in the methylation step of DNJ alkaloid biosynthesis in mulberry leaves, which is a breakthrough in the analysis of DNJ alkaloid biosynthetic pathway. It is worth mentioning that the novel MaMT1, annotated as serine hydroxymethyltransferase, could rely on SAM to perform C-methyltransferase function. Therefore, our findings contribute new insights into the research of DNJ alkaloid biosynthesis and C-methyltransferase family.