Molecular and in silico characterization of tryptophan decarboxylase (TDC) gene from mitragyna speciosa (Kratom)

Abstract

Mitragyna speciosa, commonly known as kratom or “ketum”, is a tropical plant belonging to the Rubiaceae (coffee) family, predominantly found in Southeast Asia, particularly in Malaysia, Indonesia and Thailand. It is widely recognized for its medicinal properties, primarily due to mitragynine, a major psychoactive compound with stimulant and analgesic effects. Mitragynine biosynthesis involves a complex metabolic pathway in which the enzyme tryptophan decarboxylase (TDC) plays a critical role by converting tryptophan into tryptamine, a key alkaloid precursor. This study aimed to identify and characterize the TDC gene from the M. speciosa using molecular techniques and bioinformatics, focusing on its gene structure, three-dimensional modelling and mechanistic interaction, particularly the protein-ligand interaction between the TDC and tryptophan. Functional analysis revealed that the region spanning amino acid positions 57 to 430 represents a conserved TDC protein domain involved in metal-ion interaction and ligand binding within a full-length protein of 506 amino acids. The TDC gene was successfully isolated using the conventional Cetyltrimethylammonium bromide (CTAB), followed by Polymerase Chain Reaction (PCR) amplification. The resulting 1,599 bp nucleotide sequence was validated via Needleman-Wunsch pairwise alignment. Phylogenetic analysis was conducted using MEGA-X, and domain prediction with INTERPRO confirmed the presence of the Pyridoxal-Dependent Decarboxylase domain, which binds pyridoxal phosphate (PLP) and is essential for catalyzing the decarboxylation of amino acids, enabling the conversion of L-tryptophan to tryptamine. Physicochemical analysis using PROTPARAM classified TDC as a polar protein with an optimum isoelectric point (pI) of 6.05. The three-dimensional structure modelling identified the AlphaFold model as the most reliable, with ERRAT quality score of 95.7983. Tunnel visualization revealed a ligand-accessible channel measuring 4.7 Å in length with a bottleneck radius of 2.25 Å, suggesting ideal path for substrate transport. Molecular docking analysis using Webina and SeamDock platforms showed that Webina provided a more stable and stronger interaction, with a binding affinity of -9.901 kcal/mol, compared to -5.4 kcal/mol for SeamDock. The protein–ligand complex was further refined using YASARA energy minimization, resulting in a reduced total potential energy of −6.688 × 10⁴ kcal/mol, indicating a stable and optimized complex structure. In conclusion, this study elucidates the functional role of TDC in the biosynthesis of tryptamine-derived alkaloids in M. speciosa. The findings provide a molecular basis for future metabolic engineering and synthetic biology studies targeting TDC to enhance the production of specific pharmaceutically important alkaloids, particularly mitragynine.