酪氨酸酶
生物化学
酪氨酸羟化酶
羟基化
酪氨酸
酶
多酚氧化酶
刺毛黧豆
生物
化学
过氧化物酶
植物
作者
G. Saranya,M.V. Jiby,K.S. Jayakumar,Padmesh P. Pillai,Chelliah Jayabaskaran
出处
期刊:Phytochemistry
[Elsevier]
日期:2020-10-01
卷期号:178: 112467-112467
被引量:15
标识
DOI:10.1016/j.phytochem.2020.112467
摘要
Mucuna pruriens L., commonly known as velvetbean or cow-itch, is a self-pollinated tropical legume of the family Fabaceae, known for its medicinal properties. The active principle L-DOPA extracted from the plant is a potent drug used in the treatment of Parkinson's disease. Although, it is hypothesized that a single step reaction can produce L-DOPA, the presence of optional routes makes the pathway more intricate. For instance, the catecholamine biosynthetic pathway, which leads to L-DOPA production, could occur by hydroxylation of tyrosine to L-DOPA either by polyphenol oxidase (PPO) or tyrosine hydroxylase (TH). Furthermore, Cytochrome P450 (CYP) enzymes can also cause hydroxylation of tyrosine, resulting in L-DOPA synthesis. Therefore, the present investigation was focused on validating the step, which catalyzes the synthesis of L-DOPA, at the biochemical and molecular levels. Enzyme inhibitor studies showed significant inhibition of PPO enzyme with corresponding decrease in L-DOPA synthesis while TH and CYP inhibition had no effect on L-DOPA synthesis. Activity staining of non-denaturing PAGE gel for PPO and TH showed activity only to PPO enzyme. Following in-gel assay and tryptic digestion of the excised stained gel portion, peptide recovery and LC-MS/MS analysis were performed. Degenerate primers based on peptide sequence resulted in an 800bp amplicon. The subsequent sub-cloning, RACE analysis and BLAST search resulted in the isolation of full-length PPO coding sequence of 1800 bp. Structure prediction and phylogenetic analysis of the obtained sequence revealed strong similarity to other plant PPO's like Glycine max, Vigna radiata and Vicia faba of the same family.
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