Elucidating the interaction of SCPL acyltransferase and its non-catalytic companion paralog in galloylated catechin biosynthesis via an internal epitope tagging strategy

Galloylated catechins are pivotal secondary metabolites that confer both the characteristic bitter-astringent flavor and the health benefits to tea. It is known that the co-expression of the catalytic enzyme CsSCPL4 and its non-catalytic companion paralog CsSCPL5 is essential for catalyzing the synthesis of galloylated catechins. However, how CsSCPL5 performs its companion function has not been elucidated. A key challenge is that the cleavage of C-terminal tags during post-translational processing prevents the production of functional tagged proteins, thereby impeding the investigation of the CsSCPL4-CsSCPL5 interaction mechanism. In this study, mature epitope-tagged CsSCPL5 was generated by co-expressing CsSCPL4 with CsSCPL5 bearing a Flag tag in specific internal loop regions. Co-immunoprecipitation and enzymatic assays demonstrated that the CsSCPL4-CsSCPL5 interaction occurs in both precursor and mature forms, and that the function depends on the mature CsSCPL4-CsSCPL5 heteromeric complex. Based on the heteromeric structure predicted by AlphaFold 3, site-directed mutagenesis of the interaction sites abolished enzymatic activity. Consistently, immunoblot analysis revealed that these mutations reduced precursor stability and prevented mature complex formation, indicating the importance of CsSCPL4-CsSCPL5 complex in function. Our study of the interaction between CsSCPL4 and CsSCPL5 provides new insights into the molecular mechanisms underlying the galloylated catechins biosynthetic pathway in plants.