Enzymatic Synthesis of 3-Deoxy- d -manno-octulosonate 8-Phosphate, 3-Deoxy- d -altro-octulosonate 8-Phosphate, 3,5-Dideoxy- d -gluco(manno)-octulosonate 8-Phosphate by 3-Deoxy- d -arabino-heptulosonate 7-Phosphate Synthase

The phenylalanine-sensitive 3-deoxy- d - arabino -heptulosonate 7-phosphate (DAH 7-P) synthase (phe), a key enzyme involved in the biosynthesis of the aromatic amino acid phenylalanine, expressed by the Escherichia coli gene aro G, which catalyzes the condensation of d -erythrose 4-phosphate with phospho enol pyruvate (PEP) to give DAH 7-P, was cloned into the expression vector pT7-7 for overexpression in E. coli . Purified enzyme from this expression system was used to demonstrate that DAH 7-P synthase (phe) also catalyses the aldol-type condensation of PEP with the 5-carbon analogues d -arabinose 5-phosphate, d -ribose 5-phosphate, and 2-deoxy- d -ribose 5-phosphate to yield 3-deoxy- d - manno -octulosonate 8-phosphate, 3-deoxy- d - altro -octulosonate 8-phosphate, and 3,5-dideoxy- d - gluco(manno) -octulosonate 8-phosphate, respectively, as determined by 1 H NMR and other standard analytical methods. The kinetic parameters, K m and V max, for these reactions were determined. The 3- and 6-carbon phosphorylated monosaccharides, d, l -glyceraldehyde 3-phosphate and d -glucose 6-phosphate, as well as the nonphosphorylated 5-carbon analogues d -arabinose 5-phosphate, d -ribose 5-phosphate, and 2-deoxy- d -ribose 5-phosphate were not substrates.