Abstract
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 °C. AADC-BA showed optimal pH and temperature ranges at 7–8 and 37–45 °C, respectively. The KM and kcat values for l-Phe were 7.2 mM and 7.4 s−1, respectively, at pH 7.0 and 37 °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.
Key points
• AADC-BA showed broad substrate specificity for various aromatic amino acids.
• The substrate specificity was elucidated by in silico structural modeling.
• The synthetic potential of AADC-BA was demonstrated for the production of biogenic amines.
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References
Berry MD, Juorio AV, Li XM, Boulton AA (1996) Aromatic L-amino acid decarboxylase: a neglected and misunderstood enzyme. Neurochem Res 21(9):1075–1087. https://doi.org/10.1007/BF02532418
Christendat D, Yee A, Dharamsi A, Kluger Y, Savchenko A, Cort JR, Booth V, Mackereth CD, Saridakis V, Ekiel I, Kozlov G, Maxwell KL, Wu N, McIntosh LP, Gehring K, Kennedy MA, Davidson AR, Pai EF, Gerstein M, Edwards AM, Arrowsmith CH (2000) Structural proteomics of an archaeon. Nat Struct Biol 7(10):903–909. https://doi.org/10.1038/82823
Eliot AC, Kirsch JF (2004) Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu Rev Biochem 73:383–415. https://doi.org/10.1146/annurev.biochem.73.011303.074021
Engelman DM, Steitz TA, Goldman A (1986) Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem 15(1):321–353. https://doi.org/10.1146/annurev.bb.15.060186.001541
Facchini PJ, Huber-Allanach KL, Tari LW (2000) Plant aromatic L-amino acid decarboxylases: Evolution, biochemistry, regulation, and metabolic engineering applications. Phytochemistry 54(2):121–138. https://doi.org/10.1016/S0031-9422(00)00050-9
Han SW, Shin JS (2016) A facile method to determine intrinsic kinetic parameters of ω-transaminase displaying substrate inhibition. J Mol Catal B-Enzym 133:S500–S507. https://doi.org/10.1016/j.molcatb.2017.05.001
Han SW, Park ES, Dong JY, Shin JS (2015) Mechanism-guided engineering of ω-transaminase to accelerate reductive amination of ketones. Adv Synth Catal 357(8):1732–1740. https://doi.org/10.1002/adsc.201500211
Han SW, Kim J, Cho HS, Shin JS (2017) Active site engineering of ω-transaminase guided by docking orientation analysis and virtual activity screening. ACS Catal 7(6):3752–3762. https://doi.org/10.1021/acscatal.6b03242
Kalb D, Gressler J, Hoffmeister D (2016) Active-site engineering expands the substrate profile of the basidiomycete L-tryptophan decarboxylase CsTDC. ChemBioChem 17(2):132–136. https://doi.org/10.1002/cbic.201500438
Kezmarsky ND, Xu H, Graham DE, White RH (2005) Identification and characterization of a L-tyrosine decarboxylase in Methanocaldococcus jannaschii. Biochim Biophys Acta-Gen Subj 1722(2):175–182. https://doi.org/10.1016/j.bbagen.2004.12.003
Koyanagi T, Nakagawa A, Sakurama H, Yamamoto K, Sakurai N, Takagi Y, Minami H, Katayama T, Kumagai H (2012) Eukaryotic-type aromatic amino acid decarboxylase from the root colonizer Pseudomonas putida is highly specific for 3,4-dihydroxyphenyl-L-alanine, an allelochemical in the rhizosphere. Microbiology 158(12):2965–2974. https://doi.org/10.1099/mic.0.062463-0
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157(1):105–132. https://doi.org/10.1016/0022-2836(82)90515-0
Li T, Huo L, Pulley C, Liu A (2012) Decarboxylation mechanisms in biological system. Bioorganic Chem 43:2–14. https://doi.org/10.1016/j.bioorg.2012.03.001
Magdeldin S, Yoshida Y, Li H, Maeda Y, Yokoyama M, Enany S, Zhang Y, Xu B, Fujinaka H, Yaoita E, Sasaki S, Yamamoto T (2012) Murine colon proteome and characterization of the protein pathways. BioData Min 5(1):11. https://doi.org/10.1186/1756-0381-5-11
McDonald AD, Perkins LJ, Buller AR (2019) Facile in Vitro Biocatalytic Production of Diverse Tryptamines. ChemBioChem 20(15):1939–1944. https://doi.org/10.1002/cbic.201900069
Moreno-Arribas V, Lonvaud-Funel A (2001) Purification and characterization of tyrosine decarboxylase of Lactobacillus brevis IOEB 9809 isolated from wine. FEMS Microbiol Lett 195(1):103–107. https://doi.org/10.1016/S0378-1097(00)00559-0
Nakazawa H, Kumagai H, Yamada H (1981) Aromatic L-amino acid decarboxylase from Micrococcus percitreus purification, crystallization and properties. Agric Biol Chem 45(11):2543–2552. https://doi.org/10.1271/bbb1961.45.2543
Oliveira EF, Cerqueira NM, Fernandes PA, Ramos MJ (2011) Mechanism of formation of the internal aldimine in pyridoxal 5′-phosphate-dependent enzymes. J Am Chem Soc 133(39):15496–15505. https://doi.org/10.1021/ja204229m
Park ES, Shin JS (2011) Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity. Enzyme Microb Technol 49(4):380–387. https://doi.org/10.1016/j.enzmictec.2011.06.019
Park ES, Dong JY, Shin JS (2014a) Active site model of (R)-selective ω-transaminase and its application to the production of D-amino acids. Appl Microbiol Biotechnol 98(2):651–660. https://doi.org/10.1007/s00253-013-4846-5
Park ES, Park SR, Han SW, Dong JY, Shin JS (2014b) Structural determinants for the non-canonical substrate specificity of the ω-transaminase from Paracoccus denitrificans. Adv Synth Catal 356(1):212–220. https://doi.org/10.1002/adsc.201300786
Rath A, Glibowicka M, Nadeau VG, Chen G, Deber CM (2009) Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proc Natl Acad Sci USA 106(6):1760–1765. https://doi.org/10.1073/pnas.0813167106
Sáenz-De-Miera LE, Ayala FJ (2004) Complex evolution of orthologous and paralogous decarboxylase genes. J Evol Biol 17(1):55–66. https://doi.org/10.1046/j.1420-9101.2003.00652.x
Shirai A, Matsuyama A, Yashiroda Y, Hashimoto A, Kawamura Y, Arai R, Komatsu Y, Horinouchi S, Yoshida M (2008) Global analysis of gel mobility of proteins and its use in target identification. J Biol Chem 283(16):10745–10752. https://doi.org/10.1074/jbc.M709211200
Torrens-Spence MP, Lazear M, von Guggenberg R, Ding H, Li J (2014) Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases. Phytochemistry 106:37–43. https://doi.org/10.1016/j.phytochem.2014.07.007
Williams BB, Van Benschoten AH, Cimermancic P, Donia Mohamed S, Zimmermann M, Taketani M, Ishihara A, Kashyap Purna C, Fraser James S, Fischbach Michael A (2014) Discovery and characterization of gut microbiota decarboxylases that can produce the neurotransmitter tryptamine. Cell Host & Microbe 16(4):495–503. https://doi.org/10.1016/j.chom.2014.09.001
Yuwen L, Zhang F-L, Chen Q-H, Lin S-J, Zhao Y-L, Li Z-Y (2013) The role of aromatic L-amino acid decarboxylase in bacillamide C biosynthesis by Bacillus atrophaeus C89. Sci Rep 3(1):1753. https://doi.org/10.1038/srep01753
Zhu MY, Juorio AV (1995) Aromatic L-amino acid decarboxylase: biological characterization and functional role. Gen Pharmacol 26(4):681–696. https://doi.org/10.1016/0306-3623(94)00223-A
Funding
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2019R1F1A1062845). S.-W. Han was financially supported by Initiative for Biological Function & Systems under the BK21 PLUS program of Korean Ministry of Education.
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YC, SH, JK, and YJ performed experiments. SH contributed to data analysis. JS designed, supervised, and coordinated the study. JS wrote the manuscript. SH contributed to manuscript editing. All authors read and approved the final manuscript.
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Choi, Y., Han, SW., Kim, JS. et al. Biochemical characterization and synthetic application of aromatic l-amino acid decarboxylase from Bacillus atrophaeus. Appl Microbiol Biotechnol 105, 2775–2785 (2021). https://doi.org/10.1007/s00253-021-11122-3
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DOI: https://doi.org/10.1007/s00253-021-11122-3