title: Withania coagulans tryptophan decarboxylase gene cloning, heterologous expression, and catalytic characteristics of the recombinant enzyme.
abstract: Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5'-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher Kmvalue for tryptophan than for pyridoxal 5'-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, India.
authors.forename: Jyoti Singh
authors.initials: JS
authors.lastname: Jadaun
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, India.
authors.forename: Neelam Singh
authors.initials: NS
authors.lastname: Sangwan
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, India.
authors.forename: Lokesh Kumar
authors.initials: LK
authors.lastname: Narnoliya
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, India.
authors.forename: Sandhya
authors.initials: S
authors.lastname: Tripathi
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, India. sangwan.lab@gmail.com.|Center of Innovative and Applied Bioprocessing (CIAB), C-127, Phase-8, Industrial Area, S.A.S. Nagar, Mohali, 160071, Punjab, India. sangwan.lab@gmail.com.
authors.forename: Rajender Singh
authors.initials: RS
authors.lastname: Sangwan
mesh_terms: D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D003001:Cloning, Molecular; D019295:Computational Biology; D004795:Enzyme Stability / Q000187:drug effects; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D010802:Phylogeny; D018520:Plant Shoots / Q000187:drug effects / Q000254:growth & development; D011994:Recombinant Proteins / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature; D032341:Withania / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D001142:Aromatic-L-Amino-Acid Decarboxylases
keywords: Ashwagandha; Serotonin; Tryptamine; Tryptophan; Tryptophan decarboxylase; Withanamides; Withania