Purification of thymidine-diphospho-D-glucose 4,6-dehydratase from an erythromycin-producing strain of Saccharopolyspora erythraea by high resolution liquid chromatography.

TDP-D-glucose 4,6-dehydratase was purified from Saccharopolyspora erythraea, the producer of the macrolide antibiotic erythromycin A, by a high resolution chromatographic method that exploited the difference in the behavior of the protein on anionic exchange chromatography in Tris/HCl or phosphate buffers. By this method, the enzyme was purified approximately 900-fold by two anionic exchange steps to more than 90% homogeneity. It was further purified to apparent homogeneity by hydrophobic interaction chromatography. The enzyme is a homodimer of Mr 36,000 subunits, is highly specific for TDP-D-glucose, requires NAD+ as cofactor, and shows a K'm of 34 microM and V'max of 26 mumol h-1 mg-1 of protein for TDP-D-glucose. TDP and TTP strongly inhibit the enzyme at 2 mM. The maximal TDP-D-glucose 4,6-dehydratase activity coincides with the time of erythromycin production, suggesting that this enzyme is involved in antibiotic biosynthesis.