Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I

Abstract

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide de ned by its repeating disaccharide backbone structure of [4)-α-d-GalA-(1,2)-α-l-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identi ed, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown.Here, a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to re ect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into polymeric product. RGGAT1 is a member of a new GT family categorized as GTx that is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan (HG). RGGAT1 has a predicted GT-A fold structure, but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type, suggesting that RG-I biosynthesis has functionally diverged from the mechanisms used for synthesis of HG and other plant cell wall polysaccharides.

Daily glycome and transcriptome profiling reveals polysaccharide structures and glycosyltransferases critical for cotton fiber growth

“…Galacturonosyltransferases (RG-I:GalATs), RG-I:rhamnosyltransferases (RRTs), and O-acetyltransferases (TBR/TBL) are required to synthesize the primary backbone of RG-I (Fig. 2) (Atmodjo et al 2013; Amos et al 2022). β-1,4-galactan synthases (GALSs) synthesizes β-1,4-galactan secondary backbone of RG-I.…”

A putative rhamnogalacturonan-II CMP-β-Kdo transferase identified using CRISPR/Cas9 gene edited callus to circumvent embryo lethality

“…Our attempts to express RCKT1 using the Escherichia coli Origami B strain was not successful. The Human Embryonic Kidney (HEK293) cell system 61 , provides an alternative technology as it has been used to produce several plant GTs 62–68 . However, an equally large challenge arises from the instability of CMP-β-Kdo, the donor substrate for the RCKT1 enzyme.…”

“…Our own attempt to express RCKT1 using the Escherichia coli Origami B strain was also unsuccessful. Nonetheless, the recent use of the Human Embryonic Kidney (HEK293) cell system 62 , has shown some promise for the production of soluble plant GTs [63][64][65][66][67][68][69] . However, a greater challenge arises from the inherent instability of CMP-β-Kdo, the essential donor substrate for the RCKT1 enzyme.…”

The plant cell wall—dynamic, strong, and adaptable—is a natural shapeshifter

“…The identification of the biosynthetic RG-I backbone rhamnosyltransferase (RRT) (GT106) ( Takenaka et al 2018 ) and galacturonosyltransferase (RGGAT) (GT116) ( Amos et al 2022 ) families that function together to synthesize the RG-I rhamnogalacturonan backbone ( Fig. 6B ) opens the door to in vitro synthesis of complex pectic heteroglycans and pectic AGP glycoconjugates such as APAP1 ( Tan et al 2013 ) and RG-I-AGP ( Tan et al 2023b ) ( Fig.…”