“…[46] The functional characterization of sugar-modifying O-methyltransferases has relied upon in vivo genetics/heterologous expression (as in studies relating to avermectin, [47,48] mithramycin, [49] chromomycin, [50] clorobiocin, [51,52] and avilamycin [53] ) or in vitro biochemical characterization (as in studies toward understanding oleandomycin, [34] tylosin, [35,36] and elloramycin [54] biosynthesis), and, with one exception, the avermectin TDP-olivosyl-3-Omethyltransferase, [47,48] all act post-glycosyltransfer. Consistent with previous in vivo studies, the present RebM biochemical and kinetic characterization confirms C-4'-O-methylation as the last step in the biosynthesis of 1 and places RebM among the "norm" for sugar-O-methyltransferases in the context of both timing of the methylation event as well as catalytic efficiency.…”