KDO-8-phosphate

Compound 75 served as a model for a study of the TBA reactivity of 5-O-substituted KDO derivatives17 (compare Section 11,1). A synthesis of KDO 8-phosphate (2), which occurs in Nature as the product of the KDO 8-phosphate synthetase reaction9 (Scheme 1 compare Section V,2 and 3), was performed by Charon and Szabo.113 2-O-Benzyl-D-... 

The reaction catalyzed by KDO 8-phosphate synthetase (reaction 2, Scheme 35) was first observed by Levin and Racker9 in extracts from Pseudomonas aeruginosa (see Scheme 1), and later by Ghalambor and Heath29 in extracts from Escherichia coli 0111 B4 and J-5. In the initial experiments of Levin and Racker,135 the fate of D-ribose 5-phosphate in crude bacterial extracts was studied, and the KDO 8-phosphate discovered by the authors is really derived from D-ribose 5-phosphate by three, sequential, enzyme-catalyzed reactions (see Scheme 36). 

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... 

Figure 4. Synthesis of KDO-8-phosphate. HK, hexokinase PK, pyruvate kinase.

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. 

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... 

TABLE IV. Specificity of KDO-8-phosphate Phosphatase on Phosphorylated Compounds. 

CMP-KDO synthetase (cytidine-5 -triphosphate cytidine-5 -mon-ophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase), the next enzyme in the pathway, catalyzes the formation of the nucleotide sugar, CMP-KDO from CTP and KDO. This enzyme was first studied by Ghalambor and Heath (IT). We have purified this enzyme to homogeneity (27). T i apparent K values for CTP nd KDO in the presence of 10 mM Mg were determined to be 2 x 10 M and 2.9 x 10 M, respectively. The enzy tic reaction was dependent upon the addition of CTP, KDO and Mg but did not require a reducing agent. The formation of CMP-KDO was not inhibited by the addition of CDP, CMP, KDO-8-phosphate or N-acetylneuraminic acid to the complete reaction mixture. In agreement with Ghalambor and Health (17), neither KDO-8-phosphate nor N-acetylneuraminic acid could substitute for KDO in the reaction mixture. Pyrophosphate, one of the end products, is a weak inhibitor of the reaction with an apparent Ijq value of 5.0 mM. The addition of CMP,CD or any of the other mono- or di-nucleotides did not inhibit the reaction. 

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... 

Development of the chemical synthesis of ulosonic acids which mimics the enzymatic condensation is an interesting target [70]. As a model a reaction leading to KDO 8-phosphate (22) has been evaluated. In this reaction mediated by KDO 8-P synthetase D-arabinose 5-phosphate (A5P, 21) is reacted with phosphoenolpyruvate (PEP) (Scheme 7) [71]. 

The active site of KDO 8-phosphate synthase complexed to its natural substrate and to an inhibitor has been identified by use of rotational-echo doubleresonance (REDOR) solid state NMR spectroscopy. ... 

The 5-0-a-L-rhamnopyranosyl derivative of KDO and KDO 7-(2-aminoethylphosphate) have both been isolated from the inner core region of the lipopolysaccharide (LPS) of E.coli K12. The syntheses of KDO-7-phosphate and 7-(2-acetamidoethyl phosphate) have been carried out by phosphorylation of an appropriately-protected derivative. It has been shown that in solution KDO 8-phosphate exists as a mixture of a-pyranose (66%), P-pyranose (3%), a-furanose (19%) and P-furanose (12%) forms. By use of the two isomeric 2-deoxy analogues of KDO 8-phosphate it was shown that KDO 8-phosphate phosphatase is speciflc for the a-pyranose form of the substrate, so that a mutarotation must be involved before the next stage in the formation of LPS, since CMP-KDO synthetase is specific for the P-pyranose form of KDO. 0 Other references to 2-deoxy-KDO and 2-deoxy-NeuNAc can be found in Section 1 of this Chapter. 

A multigram enzymic synthesis of KDO-8-phosphate has been devised D-arablnose-5-phosphate is prepared by hexokinase action on the sugar, and is then condensed with phosphoenolpyruvate using KDO-8-phosphate synthetase. The ATP required for the hexokinase reaction is regenerated from phosphoenolpyruvate using pyruvate kinase.22... 

In studies on the mechanism and stereochemistry of Kdo-8-phosphate synthase, use of stereospecifically fluorinated analogues of phosphoenolpyruvate led to the conclusion that the si-face of phosphoenolpyruvate was involved in bonding to D-arabinose-S-phosphate thus the fluorinated analogue 52 was produced from (E)-3-fluoro-phosphoenolpyruvate specifically deuterated materials were also used. The reaction shown in Scheme 9 was the key to the synthesis of the phosphonate 53 this proved to be a competitive inhibitor of Kdo-8-phosphate... 

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