Stereochemistry of 3-deoxyoctulosonate 8-phosphate synthase

(Z)- and (E)-[3-2H]phosphoenolpyruvate were prepared chemically by the reductive deuteration of (Z)- and (E)-3-bromophosphoenolpyruvate, respectively, and were converted into 3-deoxyoctulosonate 8-phosphates deuterated at the C-3 position by incubation with unlabeled D-arabinose 5-phosphate in the presence of the enzyme, 3-deoxyoctulosonate 8-phosphate synthase (EC4.1.2.16) purified from Escherichia coli K-12 containing the plasmid pMW101. Analysis of the stereochemistry of the two 3-deoxyoctulosonate 8-phosphates deuterated at the C-3 position by 1H NMR showed that the (Z)-[3-2H]phosphoenolpyruvate had produced [3-2H]-3-deoxyoctulosonate 8-phosphate of predominantly the 3S configuration and that the E isomer had given predominantly (3R)-[3-2H]-3-deoxyoctulosonate 8-phosphate. The 3-deoxyoctulosonate 8-phosphate synthase reaction is therefore stereospecific with respect to the C-3 of phosphoenolpyruvate. The results indicate a si face attack from the C-3 of phosphoenolpyruvate, a result identical to that reported for 3-deoxyheptulosonate 7-phosphate synthase (EC 4.1.2.15), an enzyme catalyzing an identical aldol-type condensation, except that it takes place between phosphoenolpyruvate and D-erythrose 4-phosphate. The stereochemistry with respect to the face of the carbonyl of the attacked aldehyde, in both 3-deoxyoctulosonate 8-phosphate synthase and 3-deoxyheptulosonate 7-phosphate synthase, is re. On the basis of the results of the studies reported herein, the presence of a transient methyl group at the C-3 of phosphoenolpyruvate as part of the reaction mechanism seems unlikely.