期刊
JOURNAL OF ADVANCED RESEARCH
卷 21, 期 -, 页码 57-64出版社
ELSEVIER
DOI: 10.1016/j.jare.2019.10.003
关键词
Coumarin; Bergaptol O-methyltransferase; Rational design; Depigmentation disorder
资金
- China Postdoctoral Science Foundation [2016M601922, 2018T110577]
- Natural Science Fund in Jiangsu Province [BK20170736]
- National Natural Science Foundation of China [81430092, 81703637, 31970596]
- Open Project of State Key Laboratory of Natural Medicines [SKLNMKF201708]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT_15R63]
- 111 Project from the Ministry of Education of China and the State Administration of Foreign Export Affairs of China [B18056]
Bergapten has long been used in combination with ultraviolet A irradiation to treat depigmentation disorder. However, extremely low bergapten contents in plants and difficulties in synthesizing bergapten have limited its application. Here, we developed an alternative bergapten-production method. We first determined the crystal structures of bergaptol O-methyltransferase from Peucedanum praeruptorum (PpBMT) and the ternary PpBMT-S-adenosyl-L-homocysteine (SAH)-bergaptol complex to identify key residues involved in bergaptol binding. Then, structure-based protein engineering was performed to obtain PpBMT mutants with improved catalytic activity towards bergaptol. Subsequently, a highactivity mutant was used to produce bergapten for pharmacological-activity analysis. Key PpBMT amino acids involved in bergaptol binding and substrate specificity were identified, such as Asp226, Asp246, Ser265, and Va1320. Site-directed mutagenesis and biochemical analysis revealed that the V320I mutant efficiently transformed bergaptol to produce bergapten. Pharmacological-activity analysis indicated that bergapten positively affected hair pigmentation in mice and improved pigmentation levels in zebrafish embryos. This report provides the first description of the catalytic mechanism of coumarins-specific 0methyltransferase. The high-activity V3201 mutant protein could be used in metabolic engineering to produce bergapten in order to treat depigmentation disorder. This structure-function study provides an alternative synthesis method and important advances for treating depigmentation disorders. (C) 2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
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