Glycoside‐specific glycosyltransferases catalyze regio‐selective sequential glucosylations for a sesame lignan, sesaminol triglucoside

Abstract

Summary Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid‐related plant specialized metabolites. (+)‐Sesamin and (+)‐sesamolin are major hydrophobic lignans, whereas (+)‐sesaminol primarily accumulates as a water‐soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6‐O‐glucosidic linkages [i.e. (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→2)‐O‐β‐d‐glucoside‐(1→6)‐O‐β‐d‐glucoside]. We previously reported that the 2‐O‐glucosylation of (+)‐sesaminol aglycon and β1→6‐O‐glucosylation of (+)‐sesaminol 2‐O‐β‐d‐glucoside (SMG) are mediated by UDP‐sugar‐dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2‐O‐glucosylation of SMG and (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→6)‐O‐β‐d‐glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside‐specific glycosyltransferases (GGTs) and co‐ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG‐deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar–sugar β1→6‐O‐glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two‐hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane‐associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane‐bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β‐O‐glucosylation sequence of STG biosynthesis is β1→2‐O‐glucosylation of SMG by UGT94AG1 followed by UGT94AA2‐mediated β1→6‐O‐glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio‐selectivity of sequential glucosylations catalyzed by GGTs.

Lignans of Sesame (Sesamum indicum L.): A Comprehensive Review

“…The first two glucosylation steps of (+)-sesaminol were previously identified in S. indicum [247]. Only recently, the last missing step was discovered by Ono and coworkers [248]. The first step is the 2-O-glycosylation of (+)-sesaminol to produce sesaminol monoglucoside (SMG) by the enzyme UGT71A9.…”

“…Homologs of UGT71A9 were found in S. radiatum (UGT71A10) and S. alatum (UGT71A8) [247], evidencing the conservation of these steps in Sesamum spp. The glucosylation of SMG leads to diglucoside SDG(β1→6) [(+)-sesaminol 2-O-β-D-glucosyl-(1→6)-O-β-D-glucoside] or SDG(β1→2) through consecutive glucosylation of the 6 or 2 -hydroxyl group of the sugar moiety by the enzyme UGT94D1 or UGT94AG1, respectively (Figure 9) [248]. Until recently, it was not known which glucosylation step of SMG occurs first, β1→6 or β1→2 glucosylation.…”

“…Until recently, it was not known which glucosylation step of SMG occurs first, β1→6 or β1→2 glucosylation. Ono and coworkers [248] showed that β1→2 followed by β1→6 glucosylation is the major pathway. With this discovery, the missing last step for STG biosynthesis was established.…”

Tandem UGT71B5s Catalyze Lignan Glycosylation in Isatis indigotica With Substrates Promiscuity

“…Matairesinol can be continuely converted into (−)-deoxypodophyllotoxin by cytochrome P450s (CYPs), O-methyltransferases (OMTs), and 2-oxoglutarate/Fe(II)dependent dioxygenase (2-ODD), and finally hydroxylated by CYPs to produce (−)-4'-desmethyl-epipodophyllotoxin in S. hexandrum (Lau and Sattely, 2015). In another lignan biosynthesis pathway that mainly occurs in the seeds of S. indicum, pinoresinol can be catalyzed by CYP81Q1 to sequentially generate piperitol and sesamin, then sesamin can be converted into sesamolin and sesaminol (Murata et al, 2017;Ono et al, 2020). In Forsythia suspensa, after the transformation of coniferyl alcohol into epipinoresinol, phillygenin is produced by OMT (Ono et al, 2010).…”

(+)‐Sesamin‐oxidising CYP92B14 shapes specialised lignan metabolism in sesame

“…(+)‐Sesaminol, however, is generated either through ORA or by direct oxidation at the C6 position of the aromatic ring (Figure 1; Murata et al ., 2017). Lastly, (+)‐sesaminol is glucosylated sequentially through reactions catalysed by the UDP‐glucosyltransferases (UGT) UGT71A9, UGT94D1, UGT94AA2 and UGT94AG1, thereby accumulating as a water‐soluble sesaminol triglucoside (STG; Noguchi et al ., 2008; Ono et al ., 2020).…”