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-alpha-D-ribo-hexopyran-3-ulose (3) is formed by keto-enol tautomerization during the isolation procedure from initially formed dTDP-6-deoxy-alpha-D-xylo-hexopyran-4-ulos (2). dTDP-3-deoxyglucose (4) and dTDP-3-azido-3-deoxyglucose (6) were substrates and showed Michaelis-Menten kinetics (4: K-M = 200 mu M and V-max = 130 mu mol/h mg; 6: K-M = 300 mu M and V-max = 90 mu mol/h mg). In 100-mg-scale experiments, both non-natural substrates gave the respective 6-deoxy-4-keto compounds, dTDP-3,6-dideoxy-alpha-D-erythro-hexopyran-4-ulose (5) and dTDP-3-azido-3,6-dideoxy-alpha-D-xylo-hexopyran-4-ulose (7), in yields ranging from 24 to 40%. (C) 1996 Elsevier Science Ltd.