Abstract
Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.
Highlights
Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall
The co-immunoprecipitation of galacturonosyltransferase 1 (GAUT1) with GAUT7 from A. thaliana solubilized membranes revealed that GAUT1 and GAUT7 function as a GAUT1:GAUT7 heterocomplex covalently linked by disulfide bonds [15]
The fusion tags were composed of a signal sequence, His8 tag, AviTag, superfolder GFP, and tobacco etch virus (TEV) protease recognition site followed by the respective GAUT domains using a strategy previously employed for the expression of a large library of mammalian glycosylation enzyme expression constructs [32]
Summary
Atwo-phase model for the non-processive biosynthesis of homogalacturonan polysaccharides by the GAUT1:GAUT7 complex. GAUT1:GAUT7 synthesized high-molecularweight polymeric HG (>100 kDa) in a substrate concentration– dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. HG is a linear homopolymer of 1,4-linked ␣-D-galactopyranosyluronic acid (GalA) that may be partially methylesterified at O-6 and acetylated at O-2 and O-3 It is the simplest and most abundant glycan in the family of cell wall polysaccharides known as pectins, which include HG, rhamnogalacturonan I and rhamnogalacturonan II [1]. The in vitro synthesis of HG or homogalacturonan:galacturonosyltransferase (HG:GalAT) activity was mapped to GAUT1 following MS sequencing of Arabidopsis thaliana solubilized membrane proteins [1, 14]
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