One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate.
An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.
Institute of Molecular Biology and Genetics and School of Chemical Engineering, Seoul National University, Seoul 151-742, South Korea.
Jongmin
J
Oh
Sun-Gu
SG
Lee
Byung-Gee
BG
Kim
Jae Kyung
JK
Sohng
Kwangkyoung
K
Liou
Hei Chan
HC
Lee
D000084:Acetate Kinase / Q000737:chemistry*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005947:Glucose / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D005958:Glucosephosphates / Q000737:chemistry*; D006836:Hydro-Lyases / Q000737:chemistry*; D015636:Magnesium Chloride; D008359:Mannose-6-Phosphate Isomerase / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000737:chemistry; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry*; D009713:Nucleotidyltransferases / Q000737:chemistry*; D010755:Organophosphates / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013938:Thymidine Monophosphate / Q000737:chemistry*; D013942:Thymine Nucleotides / Q000138:chemical synthesis*
D005958:Glucosephosphates; D009097:Multienzyme Complexes; D010755:Organophosphates; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C112447:dTDP-6-deoxy-4-ketoglucose; D015636:Magnesium Chloride; D013938:Thymidine Monophosphate; C011632:acetyl phosphate; C031590:glucose-1-phosphate; D000084:Acetate Kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase; D009713:Nucleotidyltransferases; C085243:dTDP-D-glucose synthase; D006836:Hydro-Lyases; C022771:dTDPglucose 4,6-dehydratase; D008359:Mannose-6-Phosphate Isomerase; D005947:Glucose