Purification and characterization of TDP-D-glucose 4,6-dehydratase from anthracycline-producing streptomycetes.
TDP-D-glucose 4,6-dehydratase, which converts TDP-D-glucose to TDP-D-4-keto-6-deoxyglucose, was purified to near-homogeneity from the daunorubicin and baumycin-producing organism Streptomyces sp. C5 (968-fold purification with a 41% recovery), and from the daunorubicin producer Streptomyces peucetius ATCC 29050 (1000-fold purification with a 37% recovery). The TDP-D-glucose 4,6-dehydratases from Streptomyces sp. C5 and S. peucetius were determined by SDS-PAGE and HPLC gel filtration to be homodimers with subunit relative molecular masses of 39,000 and 36,000, respectively. For the enzymes from both organisms, negligible activity was observed in the absence of added NAD+, or when ADP-glucose, ADP-mannose, GDP-mannose, UDP-glucose or UDP-galactose was substituted for TDP-D-glucose as substrate. For the enzyme from Streptomyces sp. C5, the K'm values for NAD+ and TDP-D-glucose were 19.2 microM and 31.3 microM, respectively. The V'max for TDP-D-glucose was 309 nmol min-1 (mg protein)-1. For the S. peucetius enzyme, the K'm values for NAD+ and TDP-D-glucose were 20.1 microM and 34.7 microM, respectively. V'max values were 180 nmol min-1 (mg protein)-1 for NAD+ and 201 nmol min-1 (mg protein)-1 for TDP-D-glucose. TDP was a good inhibitor of TDP-D-glucose 4,6-dehydratase from both organisms. The N-terminal amino acid sequence of the TDP-D-glucose 4,6-dehydratase from S. peucetius and from the erythromycin producer, Saccharopolyspora erythraea, were similar, whereas the enzyme from Streptomyces sp. C5 contained a different N-terminal amino acid sequence from either of the other two enzymes.
Thompson
M W
MW
Department of Chemistry, University of Washington, Seattle 98195.
Strohl
W R
WR
Floss
H G
HG
D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis*; D004789:Enzyme Activation; D005285:Fermentation; D006595:Hexosamines / Q000096:biosynthesis; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000494:pharmacology
D000903:Antibiotics, Antineoplastic; D006595:Hexosamines; D013942:Thymine Nucleotides; C007217:daunosamine; D013938:Thymidine Monophosphate; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; C024157:thymidine 5'-triphosphate