BIOSYNTHESIS OF STREPTOMYCIN - DTDP-DIHYDROSTREPTOSE SYNTHASE FROM STREPTOMYCES-GRISEUS AND DTDP-4-KETO-L-RHAMNOSE 3,5-EPIMERASE FROM S GRISEUS AND ESCHERICHIA-COLI-Y10

dTDP-dihydrostreptose synthase from Streptomyces griseus was purified about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration of Ultrogel AcA44. The synthase preparation was free of dTDP-4-keto-l-rhamnose 3,5-epimerase (dTDP-4-keto-6-deoxy-d-glucose 3,5-epimerase, EC 5.1.3.13) activity. A new enzyme assay using Escherichia coli Y10 as source for the epimerase and dTDP-glucose 4,6-dehydratase (dTDP-glucose 4,6-hydro-lyase, EC 4.2.1.46) was developed. In the presence of excess epimerase the apparent Km for dTDP-4-keto-6-deoxy-d-glucose was determined to be 25 μM. The molecular weight of epimerase and synthase were determined by their elution volumes from a Sephadex G-100 column to be approx. 67 000 and 32 000, respectively. The pH optimum for the epimerase was between 7.5 and 8.5. The intermediate formation of dTDP-4-keto-l-rhamnose in the epimerase reaction could be shown by detection of 6-deoxy[3H]talose after NaB3H4 reduction. Results which indicate the existence of dTDP-4-keto-l-rhamnose as a free intermediate in the epimerase reaction are reported. © 1979.