Substrate Ambiguity of 3-Deoxy- d - manno -Octulosonate 8-Phosphate Synthase from Neisseria gonorrhoeae Revisited

Abstract

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while E. coli KDO8P synthase is unable to utilize E4P as a substrate (D. L. Howe, G. Y. Sheflyan, and R. W. Woodard, unpublished data).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...

Aquifex aeolicus 3-Deoxy-d-manno-2-Octulosonic Acid 8-Phosphate Synthase: A New Class of KDO 8-P Synthase?

“…We propose that the unifying trait of Class I is the lack of a metal requirement, while that of Class II is the requirement of a divalent metal for catalysis. The KDO 8-P synthases from E. coli (Ray 1980), Salmonella typhimurium (Taylor et al 2000), Neisseria gonorrhoeae (Sheflyan et al 2000), A. aeolicus (Duewel and Woodard 2000), H. pylori J99, and C. psittaci have all been characterized. The enzymes from E. coli, S. typhimurium, and N. gonorrhoeae have all been shown to catalyze the condensation reaction in the absence of any metal cofactor.…”

“…This resulted in grouping all KDO 8-P synthases with Class I DAH 7-P synthase. The division was attributed to a difference between "narrowsubstrate specificity" and "broad-substrate specificity" KDO 8-P synthases; this division has been shown to be erroneous(Sheflyan et al 2000).…”

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

“…Kinetic parameters for the isomerization of Ru5P to A5P were determined, in triplicate, using a modified coupled Aminoff assay utilizing the 3-deoxy-D-manno-octulosonate 8-phosphate synthase (Kdo8PS) from Arabidopsis thaliana (15,16). Reaction mixtures containing final concentrations of 10 mM phosphoenolpyruvate (PEP), 0.15 mg/ml Kdo8PS, 100 mM Bis-Tris propane (pH 8.5), and 1 mM EDTA and final concentrations of Ru5P ranging from 0 to 10 mM were heated separately from mixtures of Q5LIW1 or EcKdsD⌬2CBS (100 nM final concentration) to 37°C for 3 min.…”

Reversing Evolution: Re-establishing Obligate Metal Ion Dependence in a Metal-independent KDO8P Synthase

“…A full kinetic analysis was conducted (Table 1) at pH 7.4 and 37°C, conditions that are identical with those used for the KDO8PS from the related species, Neisseria gonorrhoeae. 26 The apparent molecular mass of NmeKDO8PS was ∼30 kDa for the monomer and ∼130 kDa for the native protein, as determined by SDS-PAGE and by gel-filtration chromatography, respectively. This agrees with the calculated monomeric mass of 30,483 Da and indicates that NmeKDO8PS exists as a tetrameric protein in solution, similar to other purified KDO8PS enzymes.…”