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(Tyr112) to TTT(Phe106) and TGT(Cys174) to GGT(Gly168)—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.

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