Plant cell walls are glycan-rich extracellular matrices that fundamentally impact essential cellular processes, such as growth, adhesion, and cell shape acquisition. Understanding plant cell wall glycans requires the identification and characterization of the biosynthetic enzymes that produce these polymers. Most successful in vitro protein expression studies of plant cell wall glycosyltranferases have relied on insect, fungal/yeast, or human cell expression systems, while prokaryotic expression systems have been generally unsuccessful. Here we show that Arabidopsis FRIABLE1 (FRB1)/Rhamnogalacturonan-I Rhamnosyltransferase 8 (RRT8) can be produced in E. coli RosettaGami2 cells as N-terminal maltose binding protein fusion proteins containing C-terminal 6X-His-tags. We also report the catalytic constants of FRB1/RRT8 with apparent K M and K cat values of 226 μM and 33 min -1 for UDP-Rhamnose and 117 μM and 28.7 min -1 for RG-I, respectively. We examine the catalytic activities of mutated FRB1/RRT8 proteins based on an AlphaFold3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Enzymatic characterization of the mutated and wild type FRB1/RRT8 protein confirmed that mutation of predicted catalytic site amino acid residues resulted in 20-fold reduction in RRT activity. FRB1 also robustly polymerizes RG-I in combination with RG-I Galacturonosyltransferase 1 (RGGAT1). These results show how a robust E. coli expression system combined with AI tools can be used to increase understanding of plant cell wall glycosyltransferase structure and function.
Robichaux et al. (Sun,) studied this question.