Molecular evolution and functional divergence of CsAlaDC from CsSDC reveal key determinants for theanine biosynthesis in tea plants

Abstract

Theanine, a non-protein amino acid predominantly found in tea (Camellia sinensis), is a primary contributor to the characteristic umami and sweet taste of tea infusion and is associated with numerous health benefits. The biosynthesis of its direct precursor, ethylamine, is catalyzed by the enzyme CsAlaDC. The evolutionary origin of this enzyme and the molecular basis for its functional divergence from the serine decarboxylase CsSDC, however, remain poorly understood. In this study, through comprehensive genome-wide identification, phylogenetic, structural, and domain analyses, we demonstrate that CsAlaDC originated from CsSDC via gene duplication followed by functional specialization. Phe106 and Gly168 were identified as indispensable residues governing CsAlaDC enzymatic activity in vitro and in vivo through functional validation. Crucially, we pinpointed two specific codon substitutions-TAC(Tyr 112) to TTT(Phe 106) and TGT(Cys 174 ) to GGT(Gly 168 )-as key evolutionary mutations responsible for the functional shift from SDC to AlaDC activity. These findings elucidate the evolutionary trajectory of CsAlaDC and provide mechanistic insights into the molecular regulation of theanine biosynthesis in tea plants.

References

[1] Deng W, Ogita S, Ashihara H . Ethylamine content and theanine biosynthesis in different organs of Camellia sinensis seedlings . Z Naturforsch C J Biosci. 2009; 64: 387-90

[2] Deng W, Ogita S, Ashihara H . Distribution and biosynthesis of theanine in Theaceae plants. Plant Physiol Biochem. 2010; 48: 70-2

[3] Cheng S, Fu X, Wang X. et al. Studies on the biochemical formation pathway of the amino acid l-Theanine in tea (Camellia sinensis) and other plants . J Agric Food Chem. 2017; 65: 7210-6

[4] Wei C, Yang H, Wang S. et al. Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality . Proc Natl Acad Sci USA. 2018; 115: E4151-8

[5] Casimir J, Jadot J, Renard M . Séparation et caractérisation de la N-éthyl- γ-glutamine à partir de Xerocomus badius . BBA-Bioenergetics. 1960; 39: 462-8

[6] Li M, Liu HY, Wu DT. et al. L-Theanine: a unique functional amino acid in tea (Camellia sinensis L.) with multiple health benefits and food applications . Front Nutr. 2022; 9: 853846

[7] Xu W, Song Y, Xiao W. et al. Regulatory effects and mechanisms of L-Theanine on neurotransmitters via liver-brain Axis under a high protein diet. Mol Neurobiol. 2024; 61: 783-98

[8] Fu X, Cheng S, Liao Y. et al. Characterization of l-Theanine hydrolase in vitro and subcellular distribution of its specific product ethylamine in tea (Camellia sinensis) . J Agric Food Chem. 2020; 68: 10842-51

[9] Deng W, Ogita S, Ashihara H . Biosynthesis of theanine (γ-ethylamino-l-glutamic acid) in seedlings of Camellia sinensis . Phytochem Lett. 2008; 1: 115-9

[10] Crocomo O, Fowden L . Amino acid decarboxylases of higher plants: the formation of ethylamine. Phytochemistry. 1970; 9: 537-40

[11] Rontein D, Nishida I, Tashiro G. et al. Plants synthesize ethanolamine by direct decarboxylation of serine using a pyridoxal phosphate enzyme. J Biol Chem. 2001; 276: 35523-9

[12] Takeo T. L-alanine as a precursor of ethylamine in Camellia sinensis . Phytochemistry. 1974; 13: 1401-6

[13] Takeo T . L-alanine decarboxylase in Camellia sinensis . Phytochemistry. 1978; 17: 313-4

[14] Bai P, Wei K, Wang L. et al. Identification of a novel gene encoding the specialized alanine decarboxylase in tea (Camellia sinensis) plants. Molecules (Basel). 2019; 24: 540

[15] Bai P, Wang L, Wei K. et al. Biochemical characterization of specific alanine decarboxylase (AlaDC) and its ancestral enzyme serine decarboxylase (SDC) in tea plants (Camellia sinensis) . BMC Biotechnol. 2021; 21: 17-7

[16] Weng J . The evolutionary paths towards complexity: a metabolic perspective. New Phytol. 2014; 201: 1141-9

[17] Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways. Front Plant Sci. 2019; 10: 560

[18] Moghe GD, Last RL . Something old, something new: conserved enzymes and the evolution of novelty in plant specialized metabolism. Plant Physiol. 2015; 169: 1512-23

[19] Kawahara Y, de la Bastide M, Hamilton JP. et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice. 2013; 6: 4

[20] Komori H, Nitta Y, Ueno H. et al. Structural study reveals that Ser-354 determines substrate specificity on human histidine decarboxylase. J Biol Chem. 2012; 287: 29175-83

[21] Wang H, Zhu B, Qiao S. et al. Structure and evolution of alanine/serine decarboxylases and the engineering of theanine production. Elife. 2024; 12: RP91046

[22] Zhu B, Guo J, Dong C. et al. CsAlaDC and CsTSI work coordinately to determine theanine biosynthesis in tea plants (Camellia sinensis L.) and confer high levels of theanine accumulation in a non-tea plant . Plant Biotechnol J. 2021; 19: 2395-7

[23] Lin S, Zhang Y, Zhang S. et al. Root-specific secondary metabolism at the single-cell level: a case study of theanine metabolism and regulation in the roots of tea plants (Camellia sinensis) . elife. 2024; 13: e95891

[24] Chen T, Lin S, Chen Z. et al. Theanine, a tea-plant-specific non-proteinogenic amino acid, is involved in the regulation of lateral root development in response to nitrogen status. Hortic Res. 2023; 10: uhac267

[25] Chen K, Sun K, Ye C. et al. Metabolic engineering of theanine biosynthesis in rice endosperm to develop biofortified theaRice for benefiting human health. Plant Commun. 2025; 6: 101411

[26] Weng J, Philippe RN, Noel JP . The rise of chemodiversity in plants. Science. 2012; 336: 1667-70

[27] Panchy N, Lehti-Shiu M, Shiu S . Evolution of gene duplication in plants. Plant Physiol. 2016; 171: 2294-316

[28] Yang Y, Li Q, Li H. et al. Comprehensive identification and analysis of clusters of tandemly duplicated genes reveal their contributions to adaptive evolution of green plants. Plant J. 2025; 123: e70370

[29] Sandmeier E, Hale TI, Christen P . Multiple evolutionary origin of pyridoxal-5′-phosphate-dependent amino acid decarboxylases. Eur J Biochem. 1994; 221: 997-1002

[30] Giardina G, Montioli R, Gianni S. et al. Open conformation of human DOPA decarboxylase reveals the mechanism of PLP addition to group II decarboxylases. Proc Natl Acad Sci USA. 2011; 108: 20514-9

[31] Fenalti G, Law RHP, Buckle AM. et al. GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop. Nat Struct Mol Biol. 2007; 14: 280-6

[32] Bertoldi M, Gonsalvi M, Contestabile R. et al. Mutation of tyrosine 332 to phenylalanine converts dopa decarboxylase into a decarboxylation-dependent oxidative deaminase. J Biol Chem. 2002; 277: 36357-62

[33] Gou Y, Li T, Wang Y . Active-site oxygen accessibility and catalytic loop dynamics of plant aromatic amino acid decarboxylases from molecular simulations. Biochemistry (Easton). 2024; 63: 1980-90

[34] Agnello G, Chang LL, Lamb CM. et al. Discovery of a substrate selectivity motif in amino acid decarboxylases unveils a taurine biosynthesis pathway in prokaryotes. ACS Chem Biol. 2013; 8: 2264-71

[35] Weng J, Li Y, Mo H. et al. Assembly of an evolutionarily new pathway for alpha-pyrone biosynthesis in Arabidopsis . Science. 2012; 337: 960-4

[36] Wang Y, Jin JQ, Zhang R. et al. Association analysis of BSA-seq, BSR-seq, and RNA-seq reveals key genes involved in purple leaf formation in a tea population (Camellia sinensis) . Hortic Res. 2024; 11: uhae191

[37] Bai P. Characterization and Origination Analysis of Specific Alanine Decarboxylase (AlaDC) Gene in Tea Plants[D] . Wuhan, China: Huazhong Agricultural University; 2022

[38] Chen C, Wu Y, Li J. et al. TBtools-II: a "one for all, all for one" bioinformatics platform for biological big-data mining. Mol Plant. 2023; 16: 1733-42

[39] Maeda HA, Fernie AR, Univ. Of Wisconsin, M.W.U.S. Evolutionary history of plant metabolism. Annu Rev Plant Biol. 2021; 72: 185-216

[40] Sun Y, Shang L, Zhu Q. et al. Twenty years of plant genome sequencing: achievements and challenges. Trends Plant Sci. 2022; 27: 391-401