Structure-based tailoring of the first coumarins-specific bergaptol O-methyltransferase to synthesize bergapten for depigmentation disorder treatment

Yucheng Zhao; Nana Wang; Huali Wu; Yuanze Zhou; Chuanlong Huang; Jun Luo; Zhixiong Zeng; Lingyi Kong

doi:10.1016/j.jare.2019.10.003

Structure-based tailoring of the first coumarins-specific bergaptol O-methyltransferase to synthesize bergapten for depigmentation disorder treatment

J Adv Res. 2019 Oct 10:21:57-64. doi: 10.1016/j.jare.2019.10.003. eCollection 2020 Mar.

Authors

Yucheng Zhao¹, Nana Wang², Huali Wu³, Yuanze Zhou², Chuanlong Huang¹, Jun Luo¹, Zhixiong Zeng², Lingyi Kong¹

Affiliations

¹ Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
² National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
³ School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.

Abstract

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 high-activity 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 Val320. 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 O-methyltransferase. The high-activity V320I 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.

Keywords: Bergaptol O-methyltransferase; Coumarin; Depigmentation disorder; Rational design.