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 aroG, which catalyzes the condensation of d-erythrose 4-phosphate with phosphoenolpyruvate (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 1H 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.

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The mono/bifunctional and metallo/non-metallo properties of Bacillus subtilis DAHPS (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) have been controversial for several decades. The present study investigated the DAHPSs from both the B. subtilis parent Marburg strain and the derivative strain 168 in detail and clarified the above two long-standing questions. The DAHPSs from the parent and the derivative 168 strains have identical sequence and are both bifunctional enzymes with a CM (chorismate mutase) activity and a DAHPS activity. The parent strain expresses a second independent monofunctional CM, encoded by aroH, that is highly active, while the 168 strain expresses an aroH containing a single residue mutation (A112V) that is significantly less active thus leading to previous confusion regarding the mono/bifunctionality of DAHPS. Metal analysis showed that B. subtilis DAHPS as isolated contained iron and zinc and is inactivated by dipicolinic acid; the inactive apoenzyme can be reactivated by bivalent metal ions, indicating that the enzyme is a metalloenzyme. The enzyme-bound metal is insensitive to EDTA treatment, leading to the previous conclusion that this DAHPS does not require a metal. The enzyme displays a homotetrameric structure in solution and appears to follow Michaelis-Menten kinetics with K(m)(PEP)=139+/-11.4 microM for phosphoenolpyruvate, K(m)(E4P)=1760+/-110 microM for D-erythrose 4-phosphate, kcat=4.6+/-0.1 s(-1) for DAHPS activity and K(m)(chorismate)=850+/-97 microM, kcat=0.41+/-0.01 s(-1) for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.

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3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase (phenylalanine repressible) catalyze similar aldol-type condensations between phosphoenolpyruvate (PEP) and the monosaccharides D-arabinose 5-phosphate (A5P) and D-erythrose 4-phosphate (E4P), respectively (Fig. 1). Both enzymes from Escherichia coli are tetrameric, and although their quaternary structures are dissimilar, the monomeric subunits that comprise each enzyme are nearly superimposable (9). As one might expect from this degree of subunit similarity, the facial selectivities of both enzymes from E. coli have been shown to be re-face with respect to the phosphorylated monosaccharide and si-face with respect to PEP, resulting in an (R) configuration at the C-4 position of each product (2,4,7,8). Reports from this laboratory have shown that E. coli DAH7P synthase (encoded by aroG) is capable of utilizing arabinose 5-phosphate, ribose 5-phosphate, and 2-deoxyribose 5-phosphate to synthesize the corresponding eight carbon monosaccharides (14), while E. coli KDO8P synthase is unable to utilize E4P as a substrate (D. L. Howe, G. Y. Sheflyan, and R. W. Woodard, unpublished data). The facial selectivity of DAH7P synthase with regard to both PEP and the alternate substrates listed above is the same as with the natural substrates (16a).It has recently been reported not only that KDO8P synthase from Neisseria gonorrhoeae utilizes E4P as an alternate substrate but also that the facial selectivity of the enzyme with respect to this monosaccharide is si-face, resulting in an (S) configuration at the C-4 position of the product (16). This suggests that the enzyme from N. gonorrhoeae is distinct from all other reported KDO8P synthases, in terms of both its substrate ambiguity and its stereoselectivity. However, these unusual results were obtained using enzyme isolated directly from N. gonorrhoeae that was neither rigorously purified nor thoroughly biochemically characterized (16). The product of the enzymatic reaction, C 7 -X (original authors' designation), was neither isolated nor purified, and its stereochemistry was assigned solely on the intensity and rapid production of the chromophore produced in the classic Aminoff assay (periodate-thiobarbituric acid assay) for 3-deoxy-monosaccharides (1, 10). This assay is known to be sensitive to the stereochemistry of the hydroxyl groups of the monosaccharide (11, 18) but is also known to give false-positive results with compounds such as shikimic acid, guanosine, and adenosine, as well as with DNA (6). Since studies in our laboratory are aimed at the elucidation of the similarities and differences between KDO8P and DAH7P synthase, we were intrigued by the reported use of E4P by a KDO8P synthase and decided to further investigate this unusual finding.An open reading frame corresponding to the KDO8P synthase gene (kdsA) was retrieved via a TBLASTN search of the N. gonorrhoeae FA 1090 databank at the University of Oklahoma Advanced Center for Genome Techno...