Abstract text: Understanding plant cell wall glycan structure requires the identification and characterization of the biosynthetic enzymes that produce these polymers. In vitro protein expression studies of plant cell wall glycosyltranferases have relied on insect, fungal, 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. We also report the catalytic constants of FRB1/RRT8 with apparent Km and Kcat values of 226 µM and 33 min-1 for UDP-Rhamnose and 117 µM and 28.7 min-1 for RG-I, respectively. We also identified important residues required for catalysis based on an AlphaFold3-generated FRB1/RRT8 protein structural model with a virtually docked UDP-Rha donor. Subsequent activity measurements 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). Efforts to produce other cell wall glycosyltransferases in this system will be discussed. 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.