Fig. 1: Purification and activities of recombinant metformin and dimethylguanidine hydrolases MefH and DmgH.

A Genomic structure of the genes conferring metformin or dimethylguanidine hydrolase activity. B Reactions catalyzed by heteromeric metformin hydrolase MefH and dimethylguanidine hydrolase DmgH. C Representative Coomassie stained denaturing polyacrylamide gel of DmgH, A. niigataenis AnMefH and P. mendocina PmMefH purified by Strep-Tactin affinity chromatography after expression in the presence of 0.5 mM Ni²⁺. In all three expression systems, the first hydrolase was Strep-tagged and the second hydrolase was co-purified, indicating the formation of a heteromeric complex. D Bar chart showing the specific activities with 2 mM metformin or dimethylguanidine as substrates of the three different enzyme systems. Activities were determined as the substrate and enzyme-dependent production of urea (U) or guanylurea (GU). Guanylurea was determined by LC-MS and urea was determined by a colorimetric assay. E Metal dependency of PmMefH. PmMefH was expressed in the presence of 50 µM of the respective metal ions and purified by nickel affinity chromatography (Supplementary Fig. 5). Guanylurea production rates were determined for purified PmMefH expressed in media supplemented with the divalent cations of the indicated metals. F Urea (U) or guanylurea (GU) production at different substrate concentrations was measured to determine Michaelis constant K_M and maximal reaction rate (v_max) for DmgH (black circles), AnMefH (green squares) and PmMefH (blue triangles). Data were fitted using the Michaelis-Menten equation (R² = 0.9934; R² = 0.9946, R² = 0.9950, respectively). G Urea (U) or guanylurea (GU) production was measured at different temperatures to determine the apparent temperature optimum of MefH and DmgH. Legend as in E. For D–G, the data represent the average of triplicates and consistent results were obtained with independent preparations (n = 3, error bars, s.d.). Please note that for the temperature dependency of DmgH in G the mean of duplicates are shown and some error bars are not visible in F and G as they are smaller than the symbols used to indicate the means. Source data are provided as a Source Data file.