Novel <i>α</i> ‐KG/Fe(II)‐Dependent Dioxygenases Catalyzing C1 <i>β</i> ‐Hydroxylation and Construction of 5/7/6‐Skeleton of Highly Oxygenated Taxoids
Abstract
Abstract Here, we report the discovery and functional characterization of one novel taxane C1 β ‐hydroxylase ( Tm T1 β H), belonging to the α ‐ketoglutarate ( α ‐KG)/Fe(II)‐dependent dioxygenase family from Taxus × media cell cultures. The incubation of recombinant Tm T1 β H with 1 β ‐dehydroxybaccatin IV ( 1 ) as a substrate led to the production of a major C1‐hydroxylated product, baccatin IV ( 1a ), and a minor product, 15‐hydroxy‐11(15→1) abeo ‐baccatin IV ( 1b ), a non‐classical 5/7/6‐type taxane. Moreover, in vitro biochemical assays, molecular docking, and molecular dynamics simulation combined with site‐directed mutagenesis revealed the critical amino acid residues for Tm T1 β H catalysis. Substrate specificity investigations revealed that Tm T1 β H preferred taxoids with high oxygenation level. Notably, we have also discovered a novel specific enzyme ( Tm 576) belonging to α ‐KG/Fe(II)‐dependent dioxygenase that was able to convert 1 to 1b independently. A mechanism that the 5/7/6‐membered carbon framework arises from prototypical 6/8/6‐type taxane skeleton via radical rearrangement was proposed based on DFT calculations. More importantly, we artificially reconstructed the biosynthetic pathway of two important taxanes, baccatin IV, and baccatin VI, from GGPP in tobacco system. This work not only fully characterizes the role of C1 β ‐hydroxylase of taxoids, but also offered new insights into the formation of taxane structural diversity.
Article Details
Authors (11)
Changkang Li
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Yuxin Wang
Department of Chemistry
Xinxin Yin
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Xincheng Sun
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Songyang Sui
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Jimei Liu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Ridao Chen
Division of Chemical Biology & Medicinal Chemistry, College of Pharmacy
Kebo Xie
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Dawei Chen
State Key Laboratory of Chemo and Biosensing, College of Chemistry and Chemical Engineering, Advanced Catalytic Engineering Research Center of the Ministry of Education
Yaotian Han
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China
Jungui Dai
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, NHC Key Laboratory of Biosynthesis of Natural Products, and Beijing Key Laboratory of Non‐Clinical Drug Metabolism and PK/PD Study, Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China