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 purfied about 50-fold by removal of protein with polyethyleneimine, (NH4)2SO4 fractionation and gel filtration on 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 microM. 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-6-rhamnose as a free intermediate in the epimerase reaction are reported.
D002238:Carbohydrate Epimerases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009702:Nucleoside Diphosphate Sugars; D012210:Rhamnose / Q000031:analogs & derivatives; D013305:Streptomyces griseus / Q000201:enzymology*; D013307:Streptomycin / Q000096:biosynthesis*; D013401:Sugar Alcohol Dehydrogenases
D009702:Nucleoside Diphosphate Sugars; D013401:Sugar Alcohol Dehydrogenases; C020091:dTDP-dihydrostreptose synthase; D002238:Carbohydrate Epimerases; D012210:Rhamnose; D013307:Streptomycin