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Cloning, characterization, and enzymatic identification of a new tryptophan decarboxylase from Ophiorrhiza pumila
Author(s) -
You Dawei,
Feng Yue,
Wang Can,
Sun Chengtao,
Wang Yao,
Zhao Degang,
Kai Guoyin
Publication year - 2021
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1935
Subject(s) - tryptophan , biochemistry , aromatic l amino acid decarboxylase , cofactor , pyridoxal phosphate , tryptamine , enzyme , biosynthesis , escherichia coli , amino acid , biology , aromatic amino acids , chemistry , stereochemistry , gene
Abstract Tryptophan decarboxylase (TDC, EC 4.1.1.28) catalyzes tryptophan decarboxylation to form tryptamine through the cofactor pyridoxal‐5′‐phosphate (PLP), a crucial stage in the production of the terpenoid indole alkaloids like camptothecin (CPT). A new gene encoding TDC was identified from the CPT‐producing plant Ophiorrhiza pumila by transcriptome analysis, termed OpTDC2. It contained a 1,536 bp open reading frame that encodes a 511 amino acid protein with a molecular mass of 57.01 kDa and an isoelectric point of 6.39. Multiple sequence alignment and phylogenetic tree analysis showed the closest similarity (85%) with the TDC from Mitragyna speciosa . Moreover, the highest expression of OpTDC2 was observed in the O. pumila root. To achieve high‐efficiency expression of OpTDC2 in Escherichia coli , we fused the TF tag onto the N‐terminal of the OpTDC2. Optimum enzymatic activity was observed at 45 °C, pH 8 and cofactor concentration of 0.1 mM. The catalytic reaction was strongly inhibited by metal ions of Cu 2+ , Zn 2+ , and Fe 2+ . The l ‐tryptophan was particularly catalyzed compared with d ‐tryptophan. Besides, the K m and k cat of the OpTDC2 were 1.08 mM and 0.78 Sec −1 , respectively. The results provided information on new functional OpTDC2 that might be used in synthetic biology for the enhanced biosynthesis of CPT in O. pumila .