Cloning, characterization and specificity of a new aromatic-L-amino-acid decarboxylases from Bufo bufo gargarizans

5-Hydroxytryptamine (5-HT) and its derivative bufotenine, which possess important physiological functions, are the primary active components in the secretions of toad parotid and skin gland. However, the biosynthetic pathway of these substances remains unclear in toads. To characterize toad's Aromatic-L-amino-acid decarboxylase (AADC), the key enzyme in the predicted 5-HT derivatives biosynthetic pathway, the full-length cDNA of AADC from Bufo bufo gargarizans ( Bbg AADC) was cloned from the parotoid gland of B. bufo gargarizans . The recombinant Bbg AADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold- Bbg AADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins . The enzymological properties of Bbg AADC were assessed, and it was determined that the optimal reaction temperature was 37 °C, the optimal pH was 8.6, and the optimum molar ratio of pyridoxal-5′-phosphate (PLP) to Bbg AADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as Bbg AADC could catalyze the production of 5-HT from 5-hydroxytryptophan (5-HTP) but not dopamine or tryptamine from levodopa or tryptophan , respectively. The Km of the recombinant protein Bbg AADC was 0.2918 mM and the maximum reaction rate (Vmax) was 1.182 μM·min −1 when 5-HTP was used as substrate. The Kcat was 0.0545 min −1 , and Kcat/Km was 0.1868 mM −1 ·min −1 . To elucidate the mechanism of Bbg AADC, molecular docking was performed with PLP and 5-HTP, or the external aldimine formed by 5-HTP and PLP. The results indicated that the active sites for Bbg AADC to bind with PLP were K303, H192, N300, A148, F309, T246, A273, and T147. W71, Y79, F80, P81, T82, H192, T246, N300, H302, F309, and R477 served as catalytically active sites for the binding of Bbg AADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of Bbg AADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.