MceD hydrolyzes apo Ent, MGE and DGE. (a) HPLC analysis of MceD-catalyzed hydrolysis of Ent. Dimer and monomer refer to the DHB-Ser dimer and DHB-Ser monomer, respectively, (b) HPLC analysis of MceD-catalyzed hydrolysis of MGE. Glc-Dimer is the monoglycosylated DHB-Ser dimer and “Monomers” refer to the Glc-DHB-Ser (elution time ~ 2 min) and DHB-Ser (elution time ~2.5 min) monomers, the former of which results from hydrolysis of the Glc-DHB-Ser dimer. (c) Kinetic traces for the MceD-catalyzed hydrolysis of Ent, lin-Ent, MGE, lin-MGE, and DGE. Corresponding kinetic parameters are listed in Table 2. All reactions were carried out at room temperature (75 mM Hepes, pH 7.5). (d) Schematic of the MceD-catalyzed hydrolysis of MGE illustrating the regioselectivity of the cut as determined by 2-D NMR. HPLC traces for assays with DGE are given in Figure S2 and the m/z data for Ent, MGE and DGE hydrolysis products are listed in Table S1.