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Abstract
Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development, 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. Progress in studying plant cell wall glycosyltransferases (GTs) has been limited by the difficulty of purifying these low abundance enzymes from native plant tissues and slow progress in developing viable heterologous expression systems. Here, we established a heterologous GAUT expression system in HEK293 cells and showed 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 (non-processive) 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. Two-phase elongation is potentially a common feature of polysaccharide synthesis and may explain why many GTs yield product distributions that deviate from the results predicted for strictly processive or distributive mechanisms of synthesis. The high yield of GAUT1:GAUT7 expressed in HEK293 cells and the thorough biochemical and kinetic characterization described here provides a standard for future studies of polysaccharide biosynthesis from plant cell walls and other organisms.