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.
Vara
J A
JA
School of Pharmacy, University of Wisconsin, Madison 53706.
Hutchinson
C R
CR
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004917:Erythromycin / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D013302:Streptomyces / Q000201:enzymology*
D004917:Erythromycin; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase