IN-VITRO SYNTHESIS OF CDP-D-ABEQUOSE USING SALMONELLA ENZYMES OF CLONED RFB GENES - PRODUCTION OF CDP-6-DEOXY-D-XYLO-4-HEXULOSE, CDP-3,6-DIDEOXY-D-XYLO-4-HEXULOSE AND CDP-3,6-DIDEOXY-D-GALACTOSE, AND ISOLATIONBY HPLC

In vitro enzymic synthesis of CDP-D-abequose, CDP-D-[U-C-14]abequose, CDP-6-deoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-D-xylo-4-hexulose wa s achieved using enzymes from cell extracts of cultures of Escherichia coli strains harbouring and expressing genes of the rfb gene cluster of Salmonella enterica LT2. From an initial synthesis step, CDP-6-deox y-D-xylo-4-hexulose was isolated after 30 min reaction, using CDP-D-gl ucose, NAD and CDP-glucose 4,6-dehydratase, followed by protein precip itation and desalting by gel chromatography (yield 90.6%). From that i ntermediate, CDP-3,6-dideoxy-D-xylo-4-hexulose was produced in a react ion using NADH and a crude extract containing the required enzymes. CD P-D-abequose synthesis was performed either in the presence of excess NADH and NADPH or using an enzymic system which regenerates low concen trations of the coenzymes. In a two-step reaction, CDP-D-glucose was f irst converted to CDP-6-deoxy-D-xylo-6-hexulose, then, following addit ion of the required coenzymes and enzymes, CDP-D-abequose was formed f rom this intermediary product in a 1-h incubation. Starting from 250 m g CDP-D-glucose, the molar yield of CDP-D-abequose after protein preci pitation and HPLC was 82%, corresponding to more than 200 mg. CDP-D-[U -C-14]abequose was synthesised from alpha-D-[(UC)-C-14]glucose 1-phosp hate and CTP using purified glucose-1-phosphate cytidylyltransferase i n a reaction preceding the later steps. GC-MS and NMR revealed that th e hexose part of the end product was 3,6-dideoxy-D-galactose (abequose ) and that the corresponding intermediates were 4-keto-6-deoxy-D-xylo- hexose and 4-keto-3,6-dideoxy-D-xylo-hexose, respectively. The synthes ized CDP-6-deoxy-D-xylo-4-hexulose exhibited the characteristic ultrav iolet light absorption at 318 nm but no corresponding absorption was f ound for CDP-3,6-dideoxy-D-xylo-4-hexulose. A HPLC technique, where th e four CDP-sugars were baseline separated, was developed and used for enzyme assays and for the analysis of synthesized products.