Withania coagulans tryptophan decarboxylase gene cloning, heterologous expression, and catalytic characteristics of the recombinant enzyme

Protoplasma
Jyoti Singh JadaunRajender Singh Sangwan

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 Km value 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 functio...Continue Reading

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Datasets Mentioned

BETA
ACN62127.1
AAB39708.1
AEQ01059.1
BAC415151.1
AEY82397.1

Methods Mentioned

BETA
PCR
electrophoresis
column chromatography
fluorescence assay

Software Mentioned

BLASTx
ConSurf
MODEL workspace
ExPASy
MEGA
MODEL
Ligand Explorer
Chimera
MEGA5
Consurf Server

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