Purification of a thymidine-diphospho-4-keto-6-deoxy-D-glucose epimerase from an erythromycin-producing strain of Saccharopolyspora erythraea.
A thymidine-diphospho-4-keto-6-deoxy-D-glucose epimerase was purified from Saccharopolyspora erythraea, the producer of the macrolide antibiotic erythromycin, by a high resolution chromatographic method that exploited the difference in behavior of the protein on ion exchange columns at pH 7.5 and 5.5. By this procedure and by hydrophobic interaction chromatography, the enzyme was purified more than 400-fold to apparent homogeneity. The epimerase is a monomer of M(r) 55,000, as determined by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The apparent Michaelis-Menten kinetic constants were determined to be K'm of 120 microM and V'max of 0.38 mumol mg-1 min-1. Southern analysis indicates that this epimerase is encoded by a gene that is not located within the known confines of the erythromycin biosynthetic gene cluster.
Jarvis
B W
BW
School of Pharmacy, University of Wisconsin, Madison 53706.
Hutchinson
C R
CR
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002238:Carbohydrate Epimerases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000032:analysis; D004917:Erythromycin / Q000096:biosynthesis*; D005813:Genes, Synthetic; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D010446:Peptide Fragments; D004917:Erythromycin; D002238:Carbohydrate Epimerases; C085762:thymidine-diphospho-4-keto-6-deoxyglucose epimerase