D-arabinose-5-phosphate

Most of the structural and biochemical work related to KDO is based on the estimation of the compound or its derivatives by the periodate-thiobarbituric acid (TBA) assay in its various modifications. Indeed, KDO (see Fig. 3) was discovered9 through the formation of a characteristic, purple, TBA chromophore (Xmax 549 nm) from its 8-phosphate (2), which is the product of the condensation of D-arabinose 5-phosphate with enolpyruvate phosphate, catalyzed by 3-deoxy-8-0-phosphonooctulosonate synthetase (EC 4.1.2.16) (see Scheme 1 and Section V,2). 

These circumstances became apparent to the authors when they attempted to study the formation of KDO 8-phosphate as catalyzed by purified bacterial extracts. These extracts did not catalyze the formation of KDO 8-phosphate from D-ribose 5-phosphate, but required D-arabinose 5-phosphate as the substrate Heath and Ghalambor29 showed that the KDO 8-phosphate synthetase reaction, observed in Pseudomonas extracts by Levin and Racker, is also catalyzed by extracts from Escherichia coli strains 0 111 B4 and J-5. Rick and Osborn136 showed that the KDO 8-phosphate synthetase from a Salmonella typhimurium mutant conditionally defective in cell-wall synthesis had a KM of 6 mM as compared to a KM of 170 pM for the enzyme from wild-type cells. 

A comprehensive study of KDO 8-phosphate synthetase has been reported by Ray.137 The author purified the enzyme 450-fold from crude extracts of Escherichia coli B cells. The synthetase has a molecular mass of 90,000 6,000 daltons and is composed of three identical subunits having an apparent molecular mass of32,000 4,000 daltons. Two pH optima were observed, one being at pH 4.0-6.0 in succinate buffer, and the other, at pH 9.0 in glycine buffer. The isoelectric point of the enzyme is 5.1. The enzyme has an apparent KM for D-arabinose 5-phosphate of 20 pM and an apparent KM for enolpyruvate phosphate of 6 pM. 

Salmonella typhimurium mutant.136-149 The defect of this mutant lies in the apparent KM (D-arabinose 5-phosphate) of its KDO 8-phosphate synthetase (compare this Section, 2). This KM increases more than 25-fold between 29 and 42°, so that the cells become increasingly dependent on exogenously supplied D-arabinose 5-phosphate as the growth temperature is raised. Cessation of LPS biosynthesis under nonper-missive conditions is accompanied by the accumulation of a KDO-de-ficient, precursor molecule.149 Lehmann150 and Rick and coworkers151 described studies directed at the isolation and chemical characterization of such lipid A precursors (for example, 133, Scheme 39). The... 

Several observations regarding this aspect have been published, and are briefly mentioned here. 5,6-Dideoxy-6-C-phosphono-D-arabino-hexofuranose (135), an isosteric phosphonate analog of D-arabinose 5-phosphate, is apparently converted, in the presence of enolpyruvate phosphate, into 3,8,9-trideoxy-9-C-phosphono-D-mcmno-2-nonulosonic acid (136) under catalysis by KDO 8-phosphate synthetase from Escherichia coli K 235. Compound 136, an isosteric phosphonate analog of KDO 8-phosphate, is a product inhibitor of the synthetase, and, by the nature of the phosphonate group, is not subject to dephosphorylation as catalyzed by KDO 8-phosphate phosphatase156 (see Scheme 40). Compound 119 (see Scheme 33) is a weak inhibitor of KDO 8-phosphate synthetase.81 KDO inhibits KDO 8-phosphate phosphatase,139 and D-ribose 5-phosphate has an inhibitory... 

D-Erythrose-4-phosphate = D-Arabinose-5-phosphate = N -Acetyl-D-mannosamin-6-phosphate... 

This enzyme [EC 2.7.1.54] catalyzes the reaction of ATP with D-arabinose to generate ADP and D-arabinose 5-phosphate. 

This enzyme [EC 5.3.1.13] catalyzes the interconversion of D-arabinose 5-phosphate to D-ribulose 5-phosphate. 

This enzyme [EC 4.1.2.16] (also known as phospho-2-dehydro-3-deoxyoctonate aldolase, phospho-2-keto-3-deoxyoctonate aldolase, and 3-deoxy-D-manno-octulo-sonic acid 8-phosphate synthetase) catalyzes the reaction of 2-dehydro-3-deoxy-D-octonate 8-phosphate and orthophosphate to produce phosphoenolpyruvate, D-arabinose 5-phosphate, and water. 

M. D. Bednarski, D. C. Crans, R. Dicosimo, E. S. Simon, P. D. Stein, and G. M. Whitesides, Synthesis of 3-deoxy-i>manno-2-octulosonate-8-phosphate (KDO-8-P) from D-arabinose Generation of D-arabinose-5-phosphate using hexokinase, Tetrahedron Lett. 29 427 (1988). 

Two of the most frequent monosaccharide components of bacterial polymers belonging to this group have been the subjects of articles in this Series. They are 3-deoxy-D-manno-2-octulosonic acid,247 a normal constituent of the core region of bacterial lipopolysaccharides that is also present in some other polymers, and N-acetylneuraminic acid,248 found in several capsular polysaccharides. Enolpyruvate phosphate serves as the precursor of the C-l-C-3 fragment of the monosaccharides, with D-arabinose 5-phosphate or 2-acetamido-2-deoxy-D-mannose 6-phosphate being an acceptor for transfer of the three-carbon unit. Characteristic, activated forms of these monosaccharides are the CMP derivatives. 

The 3-deoxy-D-ma/ino-2-octulosonic acid 8-phosphate synthase (KDO synthase or KdoS EC 4.1.2.16) is an enzyme involved in the biosynthesis of the eight-carbon sugar KDO [63], a constituent of the capsular polysaccharides (K-antigens) and outer membrane lipopolysaccharides (LPS, endotoxin) of Gram-negative bacteria [156], as well as of the cell wall of algae and a variety of plants [157], In vivo, the enzyme catalyzes the irreversible addition of 26 to D-arabinose 5-phosphate (Ara5P, 32) to form KDO 8-phosphate 33 [158]. The... 

Kdo-synthetase catalyzes the aldol addition of enolpyruvate phosphate with D-arabinose 5-phosphate (see Scheme 13), which gives 3-deoxy-r> manno-2-octulosonic add 8-phosphate (Kdo 8-phosphate). Kdo is an important component of oligosaccharides of Gram-negative bacteria. 

Scheme 13.—The Aldol Addition ofEnolpyruvate Phosphate with D-Arabinose 5-Phosphate, Catalyzed by Kdo-Synthetase.

Figure 2. Expanded pathway for the synthesis and use of KDO. The pathway initiates with D-giucose-6-phosphate, shows the branch point whereby D-ribulose-5-phosphate can be used to synthesize either D-arabinose-5-phosphate or D-ribose-5-phosphate and terminates with the transfer of KDO to the lipid A precursor. The numbers in parenthesis below the various enzymes correspond to their specific activities (nmoles per minute per...

The reactions, 1-5 respectively, are catalyzed by D-arabinose-5-phosphate isoraerase, KD0-8-phosphate synthase, KD0-8-phosphate phosphatase, CMP-KDO synthetase and KDO transferase (s). Using E. coli B, we have isolated and purified the second, third and fourth enzymes to homogeneity and studied their properties. The fifth enzyme has been partially purified by Osborn s laboratory (15) and we have not improved on its purification or further studied its properties. 

D-Arabinose-5-phosphate isomerase, the first key enzyme in the synthesis of KDO, catalyzes the interconversion of D-ribulose-5-phosphate and D-arabinose-5-phosphate. This enzyme was briefly studied by Volk (18) and later by Lim and Cohen (19). Due to the instability of the enzyme, we have only purified this enzyme 100-fold. The reversible reaction is readily monitored by measuring the formation of the keto-sugar from the aldo-sugar by the method of Dische and Borenfreund (20). The K values for D-ribulose-5-phojsphate and D-arabinose-5-phosphate are 0.9 to 1.5 and 1 to 3 x 10 M, respectively. 

TABLE I. Inhibition of D-Arabinose-5-phosphate Isomerase by Substrate Analogues... 

The second enzyme in the sequence studied was KD0-8-phosphate synthase. This enzyme was purified to homogeneity (24). This enzyme catalyzes the condensation of D-arabinose-5-phosphate and PEP to yield KD0-8-phosphate and inorganic phosphate. One can assay this irreversible reaction either by measuring the formation of KD0-8-phosphate or the release of P. (2Jj). The latter is the method of choice, since a number of analogues were found to interfer with the thiobarbituric acid assay. The enzyme has an apparent K for PEP of 6 x 10 M and an apparent K for D-arabinose-5-phospftiate of 2 x 10 M. m... 

The data in Table III indicate that the enzyme KD0-8-phos-phate synthase is more difficult to inhibit with phosphorylated substrate analogues than is D-arabinose-5-phosphate isomerase. Non-phosphorylated substrate analogues (Tables II) were also tested as inhibitors or substrates of KDO-8-phosphate synthase but as with D-arabinose-5-phosphate isomerase, these analogues were... 

The third enzyme in the pathway, KD0-8-phosphate phosphatase, has been purified to homogeneity (26). Because of its abosolute specificity, it should be a focal point for chemotherapeutic studies. jThe apparent for KD0-8-phosp te was+ etermined to be 5.8 x 10 M in the presence of 1.0 mM Co or Mg. This specific KD0-8-phosphate phosphatase was separated from enzymes, present in crude extracts, having phosphatase activity on other phosphorylated compounds by column chromatography on DGAE-Sephadex (26). Three distinct peaks of activity were detected. Fractions from each peak were pooled and the rates for the hydrolysis of five compounds were measured. Peak A possessed phosphatase activity for D-glucose-6-phosphate, D-arabinose-5-phosphate, D-ribose-5-phosphate and j-nitrophenylphosphate Peak B dephosphorylated D-arabinose-5-phosphate, D-ribose-5-phosphate and D-glucose-6-phos-phate. Peak C, which was well separated from the other two peaks, could only utilize KD0-8-phosphate as a substrate. KD0-8-phos-phate was not hydrolyzed by the phosphatases present in peaks A and B. 

In vivo measurements of lipopolysaccharide synthesis in E. coli B have indicated that two nanomoles of KDO must be synthesized per minute per mg of protein in order to meet the cellular requirement for LPS synthesis under the normal conditions of growth on glucose-minimal medium (27). We have measured the specific activities of the enzymes involved in KDO synthesis in crude extracts of E. coli B including those enzymes responsible for the synthesis of D-ribulose-5-phosphate, the precursor of D-arabinose-5-phosphate. D-Ribulose-5-phosphate is a key intermediate in carbohydrate metabolism as shown in Figure 2, since it is the direct precursor of both D-ribose-5-phosphate and D-arabinose-5-phosphate... 

D-Glucose-6-phosphate dehydrogenase, D-gluconate-6-phosphate dehydrogenase, KDO-8-phosphate synthase, KDO-8-phosphate phosphatase and CMP-KDO synthetase have been purified to homogeneity and characterized. Munson, Rasmussen and Osborn (15) have partially purified one KDO transferase. D-Arabinose-5-phosphate isomerase is very unstable and has only been purified about 100 fold. D-Ribose-5-phosphate isomerase activity is approximately 20x that... 

Measurements of the crude specific activity (mmoles of product synthesized per minute per mg of protein in the supernatant after a 50,000 x g centrifugation) of the two isomerases in E. coli indicated that the conversion of D-ribulose-5-phosphate to D-ri-bose-5-phosphate was approximately 20- to 30-fold greater than the conversion of D-ribulose-5-phosphate to D-arabinose-5-phosphate. This rate of reaction strongly pulls the reaction substrate to D-ribose-5-phosphate, since the isomerase reaction at equilibrium strongly favors the formation of the aldo-sugar over the key intermediate D-ribulose-5-phosphate. 

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