Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya, Tokyo, 156-8502, Japan.
Department of Bioscience, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya, Tokyo, 156-8502, Japan.
Plant J. 2024 May;118(3):892-904. doi: 10.1111/tpj.16644. Epub 2024 Jan 28.
The indole alkaloid gramine, 3-(dimethylaminomethyl)indole, is a defensive specialized metabolite found in some barley cultivars. In its biosynthetic process, the tryptophan (Trp) side chain is shortened by two carbon atoms to produce 3-(aminomethyl)indole (AMI), which is then methylated by N-methyltransferase (HvNMT) to produce gramine. Although side chain shortening is one of the crucial scaffold formation steps of alkaloids originating from aromatic amino acids, the gene and enzyme involved in the Trp-AMI conversion reactions are unknown. In this study, through RNA-seq analysis, 35 transcripts were shown to correlate with gramine production; among them, an uncharacterized cytochrome P450 (CYP) gene, CYP76M57, and HvNMT were identified as candidate genes for gramine production. Transgenic Arabidopsis thaliana and rice overexpressing CYP and HvNMT accumulate AMI, N-methyl-AMI, and gramine. CYP76M57, heterologously expressed in Pichia pastoris, was able to act on Trp to produce AMI. Furthermore, the amino group nitrogen of Trp was retained during the CYP76M57-catalyzed reaction, indicating that the C shortening of Trp proceeds with an unprecedented biosynthetic process, the removal of the carboxyl group and C and the rearrangement of the nitrogen atom to C. In some gramine-non-accumulating barley cultivars, arginine 104 in CYP76M57 is replaced by threonine, which abolished the catalytic activity of CYP76M57 to convert Trp into AMI. These results uncovered the missing committed enzyme of gramine biosynthesis in barley and contribute to the elucidation of the potential functions of CYPs in plants and undiscovered specialized pathways.
色胺生物碱草棉灵是一种防御性的特殊代谢物，存在于一些大麦品种中。在其生物合成过程中，色氨酸（Trp）侧链缩短两个碳原子产生 3-（氨甲基）吲哚（AMI），然后由 N-甲基转移酶（HvNMT）甲基化生成草棉灵。尽管侧链缩短是芳香族氨基酸衍生生物碱的关键支架形成步骤之一，但涉及 Trp-AMI 转化反应的基因和酶尚不清楚。在这项研究中，通过 RNA-seq 分析，发现 35 个转录本与草棉灵的产生相关；其中，一个未鉴定的细胞色素 P450（CYP）基因 CYP76M57 和 HvNMT 被鉴定为草棉灵产生的候选基因。过表达 CYP 和 HvNMT 的转基因拟南芥和水稻积累 AMI、N-甲基-AMI 和草棉灵。在巴斯德毕赤酵母中异源表达的 CYP76M57 能够作用于 Trp 生成 AMI。此外，CYP76M57 催化反应中保留了 Trp 的氨基氮，表明 Trp 的 C 缩短遵循一个前所未有的生物合成过程，即去除羧基和 C，并重新排列氮原子到 C。在一些不积累草棉灵的大麦品种中，CYP76M57 中的精氨酸 104 被苏氨酸取代，这使 CYP76M57 失去了将 Trp 转化为 AMI 的催化活性。这些结果揭示了大麦中草棉灵生物合成的缺失关键酶，并有助于阐明植物中 CYP 的潜在功能和未发现的特殊途径。
