Preparative synthesis of dTDP-L-rhamnose through combined enzymatic pathways.
dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1-phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI-MS, and NMR, showing about 95% purity.
Kang
Young-Bok
YB
School of Chemical and Biological Engineering, Seoul National University, Korea.
Yang
Yung-Hun
YH
Lee
Kwang-Won
KW
Lee
Sun-Gu
SG
Sohng
Jae Kyung
JK
Lee
Hei Chan
HC
Liou
Kwangkyoung
K
Kim
Byung-Gee
BG
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D009702:Nucleoside Diphosphate Sugars / Q000138:chemical synthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012230:Rhizobiaceae / Q000201:enzymology* / Q000235:genetics; D013942:Thymine Nucleotides / Q000138:chemical synthesis*
D001426:Bacterial Proteins; D004798:Enzymes; D009702:Nucleoside Diphosphate Sugars; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose