GlfT2 generates a heteropolysaccharide composed of alternating, regioisomeric linkages. (A) Disaccharide repeating units in heteropolysaccharides can consist of two distinct monosaccharides, as found in hyaluronic acid, or of a single monosaccharide linked with alternating, regioisomeric linkages, as found in polysialic acid or mycobacterial galactan (shown in part B). (B) GlfT2 mediates mycobacterial galactan formation by catalyzing the addition of Galf residues from UDP-Galf to an oligosaccharide–lipid conjugate (compound 1). GlfT2 has dual activities as it can generate alternating Galf-β-(1→5)-Galf and Galf-β-(1→6)-Galf linkages. (C) The synthetic acceptor substrates used in this study possess a disaccharide of Galf-β-(1→6)-Galf (compounds 2–4) or Galf-β-(1→5)-Galf (compound 5) linked to an aryl-terminated (2, 3, 5) or aliphatic (4) lipid.