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

Abstract

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 (BbgAADC) was cloned from the parotoid gland of B. bufo gargarizans. The recombinant BbgAADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold-BbgAADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins. The enzymological properties of BbgAADC 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 BbgAADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as BbgAADC 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 BbgAADC 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 BbgAADC, 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 BbgAADC 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 BbgAADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of BbgAADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.