Cloning and enzymatic function characterization of a novel tropinone reductase I (DaTRI 2) in Datura arborea
10.16438/j.0513-4870.2018-0994
- VernacularTitle:木本曼陀罗中一条新的TRI基因克隆与酶活功能鉴定
- Author:
Wei QIANG
1
;
Ke XIA
2
;
Xu-peng ZHAO
3
;
Wei FU
1
;
Jian-min MAN
1
;
Ming-sheng ZHANG
1
Author Information
1. School of Life Sciences / Resources Conservation and Germplasm Innovation in Mountainous Region Ministry of Education, Guizhou University, Guiyang 550025, China
2. Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin 541006, China
3. Guiyang University, Guiyang 550005, China
- Publication Type:Research Article
- Keywords:
tropinone reductase;
italic>Datura arborea;
tropane alkaloid;
enzymatic kinetics
- From:
Acta Pharmaceutica Sinica
2019;54(3):574-581
- CountryChina
- Language:Chinese
-
Abstract:
Tropinone reductase I (TRI) is a key branch point enzyme in the midstream of tropane alkaloids (TAs) biosynthesis pathway and represents an important target for TAs metabolic engineering, which can lead to metabolic flux of substrate tropinone to TAs. A novel TRI gene was isolated from Datura arborea, a woody resource plant, and designated as DaTRI2 (GenBank accession number is MH705164). The full-length cDNA of DaTRI2 with 1 135 bp exhibits a high sequence homology (96.8%) with DaTRI, and is predicted to encode a protein of 347 amino acids. Deduced DaTRI2 protein contain a conserved TGXXXGXG motif involved in NADPH binding, the catalytic N-S-Y-K tetrad motif and eleven amino acid residues important for binding to its substrate tropinone. The phylogenetic analysis revealed that DaTRI2 and other TRIs from Solanaceous plants belong to the same cluster and DaTRI2 exhibited closest phylogenetic proximity to TRIs from Datura. DaTRI2 was expressed in E. coli and the purified recombinant protein can catalyze both tropinone reduction and tropine oxidation with an optimum pH value of 8.0 and 9.6, respectively. When tropinone was used as the substrate, the Km and Vmax values of DaTRI2 at pH 6.4 were 210.05 μmol·L-1 and 69.6 nkat·mg-1 protein respectively, while the Km and Vmax values for tropine as the substrate were 188.03 μmol·L-1 and 114 nkat·mg-1 protein respectively, at pH 9.6. DaTRI2 transcript was most abundant in the young leaf, followed by the root. Cloning of DaTRI2 gene and biochemical analysis of recombinant DaTRI2 facilitate further research on the molecular mechanism on TAs biosynthesis in woody plants and provide a more potent candidate for TAs metabolic engineering.
