Biochemical characterization and synthetic application of aromatic l-amino acid decarboxylase from Bacillus atrophaeus

Aromatic l-amino acid decarboxylases (AADCs) are ubiquitously found in higher organisms owing to their physiological role in the synthesis of neurotransmitters and alkaloids. However, bacterial AADC has not attracted much attention because of its rather limited availability and narrow substrate range. Here, we examined the biochemical properties of AADC from Bacillus atrophaeus (AADC-BA) and assessed the synthetic feasibility of the enzyme for the preparation of monoamine neurotransmitters. AADC-BA was expressed in Escherichia coli BL21(DE3) and the purified enzyme showed a specific activity of 2.6 +/- 0.4 U/mg for 10 mM l-phenylalanine (l-Phe) at 37 degrees C. AADC-BA showed optimal pH and temperature ranges at 7-8 and 37-45 degrees C, respectively. The K-M and k(cat) values for l-Phe were 7.2 mM and 7.4 s(-1), respectively, at pH 7.0 and 37 degrees C. Comparison of the kinetic constants at different temperatures revealed that the temperature dependency of the enzyme was mainly determined by catalytic turnover rather than substrate binding. AADC-BA showed a broad substrate scope for various aromatic amino acids, including l-Phe, l-tryptophan (610% relative to l-Phe), l-tyrosine (12%), 3,4-dihydroxyphenyl-l-alanine (24%), 5-hydroxy-l-tryptophan (l-HTP, 71%), 4-chloro-l-phenylalanine (520%), and 4-nitro-l-phenylalanine (450%). Homology modeling and docking simulations were carried out and were consistent with the observed substrate specificity. To demonstrate the synthetic potential of AADC-BA, we carried out the production of serotonin by decarboxylation of L-HTP. The reaction yield of serotonin reached 98% after 1 h at the reaction conditions of 50 mM l-HTP and 4 U/mL AADC-BA. Moreover, we carried out preparative-scale decarboxylation of l-Phe (100 mM in 40-mL reaction mixture) and isolated the resulting 2-phenylethylamine (51% recovery yield). We expect that the broad substrate specificity of AADC-BA can be exploited to produce various aromatic biogenic amines.

Automated summary

The study demonstrates that AADC-BA from Bacillus atrophaeus has a broad substrate specificity, allowing for efficient synthesis of various aromatic biogenic amines, including neurotransmitters. Experimental results show that AADC-BA can be used for the production of serotonin and other substances.