Insights into acylation mechanisms: co‐expression of serine carboxypeptidase‐like acyltransferases and their non‐catalytic companion paralogs

Abstract

SUMMARY Serine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Single‐cell transcriptome atlas reveals developmental trajectories and a novel metabolic pathway of catechin esters in tea leaves

“…Two of 3 CHI s ( chalcone isomerase ), 1 of 2 F3’H s, FNS ( flavone synthase ), 2 of 2 FLS s ( flavonol synthase ) were predominantly expressed in epidermis cells of 1 L and then 3 L (Figure 6b ). Transcription of three Serine carboxypeptidase‐like acyltransferases ( SCPLs ) were rich in 1 L tissues except epidermis cells (Yao et al ., 2022 ). In addition, there are other genes that are expressed cell‐specifically in 3 L, such as C4H ( CSS0005999 ) and ANR ( CSS0024761 ) (Figure 6b ).…”

Chemical constituents of green teas processed from albino tea cultivars with white and yellow shoots

“…The pathway and key enzymes for galloylated and non-galloylated catechin biosynthesis in tea are well-understood based on recent studies ( Yao et al, 2022 , Dai et al, 2020 ; Cui et al, 2016b ; Jun, Lu, Docampo-Palacios, Wang, & Dixon, 2021 ). The non-galloylated catechins such as (–)–epicatechin, are synthesized from ( 2R , 3S , 4S )–leucocyanidin through three catalytic steps mediated by CsANS, CsANR, and CsLAR ( Jun, Lu, Docampo-Palacios, Wang, & Dixon, 2021 ).…”

“…The non-galloylated catechins such as (–)–epicatechin, are synthesized from ( 2R , 3S , 4S )–leucocyanidin through three catalytic steps mediated by CsANS, CsANR, and CsLAR ( Jun, Lu, Docampo-Palacios, Wang, & Dixon, 2021 ). The recent study showed that galloylated catechins ECG and EGCG are synthesized by CsSCPL4 and its non-catalytic paralog CsSCPL5, with the participation of β-glucogallin and non-galloylated catechins (–)–epicatechin or (–)–epigallocatechin ( Yao et al, 2022 ). It was surprising that the concentrations of non-galloylated catechins were higher in NB green teas, whereas galloylated catechins had a higher concentration in HJY green teas ( Fig.…”

Effects of Preharvest Shading on Dynamic Changes in Metabolites, Gene Expression, and Enzyme Activity of Three Tea Types during Processing

“…A PrimeScript RT kit was used to generate complementary DNA (cDNA) (Takara, Dalian, China). Table S3 presents the primer pairs for qRT-PCR, with the reference gene β-actin ( CSS0008920 ) . The manufacturer’s recommendations were followed during the thermal cycling.…”

“…Table S3 presents the primer pairs for qRT-PCR, with the reference gene β-actin (CSS0008920). 18 The manufacturer's recommendations were followed during the thermal cycling. Three independent biological replicates were guaranteed for each experiment.…”