OVERPRODUCTION AND ONE-STEP PURIFICATION OF ESCHERICHIA-COLI 3-DEOXY-D-MANNO-OCTULOSONIC ACID 8-PHOSPHATE SYNTHASE AND OXYGEN-TRANSFER STUDIES DURING CATALYSIS USING ISOTOPIC-SHIFTED HETERONUCLEAR NMR

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphhate synthase cata lyzes the condensation of D-arabinose 5-phosphate with phosphoenolpyru vate to give the unique 8-carbon acidic sugar 3-deoxy-D-manno-octuloso nic acid 8-phosphate (KDO 8-P) found only in Gramnegative bacteria and required for lipid A maturation and cellular growth. The Escherichia coli gene kdsA that encodes KDO 8-P synthase has been amplified by pol ymerase chain reaction methodologies and subcloned into the expression vector, pT7-7. A simple one-step purification yields 200 mg of homoge neous KDO 8-P synthase per liter of cell culture. [2-C-13,O-18]Phospho enolpyruvate (PEP) was prepared by first, exchange of [2-C-13]-3-bromo pyruvate with H-2(2) O-18 followed by reaction of the labeled bromopyr uvate with trimethylphosphite. The fate of the enolic oxygen in this m ultilabeled PEP, during the course of the KDO 8-P synthase-catalyzed r eaction with D-arabinose 5-phosphate, was monitored by C-13 and P-31 N MR spectroscopy. The inorganic phosphate formed during the reaction wa s further analyzed via mass spectral analysis of its trimethyl ester d erivative. The C-13 NMR spectrum of an incubation mixture of [2-C-13]P EP and D-arabinose 5-phosphate in H-2(2) O-18 in the presence of KDO 8 -P synthase was also recorded, [2-C-13]KDO 8-P was utilized to determi ne the extent of nonenzymatic incorporation of O-18 into the C-2 posit ion of KDO 8-P. The results indicate that the enolic oxygen of the PEP is recovered with the inorganic phosphate, and the C-2 oxygen of KDO 8-P originates from the solvent, H2O2.