Substrate specificity of native dTDP-d-glucose-4,6-dehydratase: Chemo-enzymatic syntheses of artificial and naturally occurring deoxy sugars

Incubation of dTDP-glucose with the enzyme dTDP-glucose-4,6-dehydratase [EC 4.2.1.46] from wild type E. coli B yielded a mixture of 3- and 4-keto-6-deoxy sugars after work-up. Model experiments with chemically synthesized methyl 6-deoxy-4-keto-glucoside (9) revealed that dTDP-6-deoxy-α-d-ribo-hexopyran-3-ulose (3) is formed by keto-enol tautomerization during the isolation procedure from initially formed dTDP-6-deoxy-α-d-xylo-hexopyran-4-ulose (2). dTDP-3-deoxyglucose (4) and dTDP-3-azido-3-deoxyglucose (6) were substrates and showed Michaelis-Menten kinetics (4: KM = 200 μM and Vmax = 130 μmol/hmg; 6: KM = 300 μM and Vmax = 90 μmol/hmg). In 100-mg-scale experiments, both non-natural substrates gave the respective 6-deoxy-4-keto compounds, dTDP-3,6-dideoxy-α-d-erythro-hexopyran-4-ulose (5) and dTDP-3-azido-3,6-dideoxy-α-d-xylo-hexopyran-4-ulose (7), in yields ranging from 24 to 40%.