[Cloning and functional study of MaCYP71BE347, a key enzyme for C-5 hydroxylation of DNJ polyhydroxy alkaloids in mulberry leaves].
摘要
As a powerful α-glucosidase inhibitor, 1-deoxynojirimycin(DNJ) polyhydroxy alkaloid is one of the main active ingredients of TCM Mori Folium in the prevention and treatment of diabetes. DNJ has four hydroxylation positions(C-3, C-4, C-5, and C-7). Our research group has previously elucidated the biosynthetic pathway of DNJ from lysine to 2-methylpiperidine and characterized two CYP450 hydroxylases involved in the C-4 position(MaCYP71BG22) and C-7 position(MaCYP82C169) hydroxylation. To further explore the key hydroxylase genes of other hydroxylation positions, this study employed molecular docking technology to screen one candidate hydroxylase gene, MaCYP71BE347, from the transcriptome data of mulberry leaves and conducted bioinformatics analysis and functional study on it. The coding sequence length of MaCYP71BE347 is 1 419 bp, encoding 472 amino acids. It is an alkaline, unstable and hydrophilic protein that is closely related to Morus notabilis and has conserved CYP450 hydroxylase motifs such as helix K, Meander, and heme binding sites. The yeast whole cell catalysis results showed that MaCYP71BE347 protein can catalyze selective hydroxylation at the C-5 position of 2-methylpiperidine to 2-methylpiperidine-5-ol and had no hydroxylation function for piperidine, 1-methylpiperidine, 2-hydroxymethylpiperidine, and 4-hydroxypiperidine. The Agrobacterium mediated mulberry genetic transformation system and virus-induced gene silencing(VIGS) transformation system confirmed the participation of MaCYP71BE347 in the biosynthesis of DNJ in mulberry leaves. This study discovered the CYP450 enzyme MaCYP71BE347, which catalyzes the C-5 hydroxylation of the piperidine ring in the biosynthesis pathway of DNJ and is a key enzyme gene element in the biosynthesis pathway of DNJ, laying the foundation for the subsequent complete analysis of the hydroxylation process of DNJ biosynthesis in mulberry leaves.