KDO synthetase

The principle of the assay procedure for CMP-KDO synthetase had been developed previously, to assay for CMP-NeuAc synthetase in ex-... 

Whereas Escherichia coli O 111 B4 contains only one, cytoplasmi-cally located, CMP-KDO synthetase activity, E. coli LP 1092 appears to contain two, chromatographically distinct, CMP-KDO synthetase activities. One of these is chromatographically similar to the enzyme from E. coli O 111 B4, whereas the other can be eluted from DEAE-cellulose at significantly lower salt concentration.85 The function, if any, of the additional CMP-KDO-synthetase activity is unknown. It has been suggested85 that the enzyme plays a role in the biosynthesis of the KDO-containing exopolysaccharide from E. coli LP 1092. 

Kundu et al.64 used MEKC conditions to assess the purity of two recombinant proteins a cytomegalovirus-CMP-KDO synthetase fusion protein expressed in E. coli and a hepatitis C viral protein expressed in CHO cells. Proteins were prepared in a 10-mM Tris-1% SDS buffer (pH 8.5) and analyzed in a 10-mM borate-100-mM SDS buffer (pH 9.5) in uncoated capillaries. The level of impurities, which varied with the method of protein production, agreed within 5% with results obtained by densitometric scanning of SDS-PAGE gels of the same materials. 

Enzymatic synthesis relying on the use of aldolases offers several advantages. As opposed to chemical aldolization, aldolases usually catalyze a stereoselective aldol reaction under mild conditions there is no need for protection of functional groups and no cofactors are required. Moreover, whereas high specificity is reported for the donor substrate, broad flexibility toward the acceptor is generally observed. Finally, aldolases herein discussed do not use phosphorylated substrates, contrary to phosphoenolpyruvate-dependent aldolases involved in vivo in the biosynthetic pathway, such as KDO synthetase or DAHP synthetase [18,19]. 

In this technique, the enzyme solution is put inside a dialysis bag which is then immersed in a solution of substrate, or cofactors. Small molecules can diffuse through the wall of the bag and react in the presence of the enzyme, while products, if also small molecules, diffuse into the outside solution, where they may be recovered. This technique has been used in syntheses with sialyl aldolase, Kdo-synthetase, the common aldolase, a mixture of hexokinase and pyruvate kinase, a-(2— 6) sialyl transferase,26 a mixture of pyruvate kinase and adenylate kinase,27 and CMP-Neu5Ac synthetase.28... 

Enzymology,29 techniques of isolation, and descriptions of a number of them. Apparently, only three have been considered for preparative chemistry, that is, aldolase, sialyl aldolase, and Kdo synthetase. However, whole cells of some strains of Escherichia coli have been used as sources of fucu-lose 1-phosphate aldolase (E.C. 4.1.2.17) or rhamnulose 1-phosphate aldolase (E.C. 4.1.2.19).30 Extraction, and concentration to a suitable degree of homogeneity, of noncommercially available aldolases are not difficult. The examination of their synthetic possibilities could be very rewarding for we already observe that the wealth of chemicals prepared with the help of aldolase and sialyl aldolase far exceeds what they make in Nature. Still, not any aldehyde, however hydrophilic, is a substrate for aldolases. 

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.

Kdo was prepared on the 38-mmol scale starting from D-arabinose, by the simultaneous operation of three enzymes in the same vessel. One is Kdo synthetase 56 hexokinase catalyzes the phosphorylation57 of D-arabinose by ATP (catalytic), and pyruvate kinase catalyzes58 the regeneration of ATP with enolpyruvate phosphate. Such systems are described in more detail in Section IV. In this preparation, enolpyruvate phosphate serves two very different purposes, acting as a source of high-energy phosphate, and as a three-carbon donor.59... 

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. 

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. 

TABLE v. Analogues of KDO Tested as Inhibitors and Substrates of CMP-KDO Synthetase... 

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... 

The requirement of the remaining enzymes, KD0-8-phosphate synthase, KD0-8-phosphate phosphatase and CMP-KDO synthetase, for their natural substrates, D-arabinose-5-phosphate + PEP, KD0-8-phosphate and KDO + CTP, respectively, was specific and the inhibition studies with substrate analogues were disappointing. Of the compounds tested as potential substrates of KD0-8-phosphate synthase, only the isosteric phosphonate analogue (Compound 11, Table III) of D-arabinose-5-phosphate was an alternate substrate (see Ref. 28). There were a number of weak competitive inhibitors of the synthase reaction (Compounds 2, 5, 6, 7, 15,and 19,Table III) the best inhibitor of the synthase reaction was 2-deoxy-2-fluoro-D-arabinonate-5-phosphate (compound 14, Table III). 

Another interesting antibiotic constmct is a peptide-conjugated form of j8-KDO. jS-KDO is an inhibitor of the CMP-KDO synthetase that is involved in lipopolysaccharide (LPS) synthesis, a major constituent of the bacterial cell wall. Antibacterial activity is thus accomplished by interference with cell wall construction. /3-KDO is incapable of membrane permeation, and therefore is not useful as an antibiotic in its native form. Hammond et al. have shown that conjugation of a /3-KDO analog to certain dipeptides resolves this problem [184] (O Fig. 14). Attachment of the /3-KDO analog to a dipeptide allows the glycopeptide constmct to permeate the bacterial membrane. Inside the cell, proteases hydrolyze the peptide and release the inhibitor, resulting ultimately in bacterial cell death. 

The research team of J. Tadanier prepared a series of C8-modified 3-deoxy-P-D-manno-2-octulosonic acid analogues as potential inhibitors of CMP-Kdo synthetase. One of the derivatives was prepared from a functionalized olefinic carbohydrate substrate by means of the Wohl-Ziegler bromination. The stereochemistry of the double bond was (Z), however, under the reaction conditions a cis-trans isomerization took place in addition to the bromination at the allylic position (no yield was reported for this step). It is worth noting that the authors did not use a radical initiator for this transformation, the reaction mixture was simply irradiated with a 150W flood lamp. Subsequently the allylic bromide was converted to an allylic azide, which was then subjected to the Staudinger reaction to obtain the corresponding allylic amine. 

Tadanier, J., Lee, C. M., Whittern, D., Wideburg, N. Synthesis of some C-8-modified 3-deoxy-]3-D-manno-2-octulosonic acid analogs as inhibitors of CMP-Kdo synthetase. Carbohydr. Res. 1990, 201, 185-207. 

Waglund, T., Luthman, K., and Orbe, M., Synthesis of C-(P-D-glycosyl) analogues of 3-deoxy-D-manno-2-octulosonic acid (Kdo) as potential inhibitors of CMP-Kdo synthetase, Carbohydr. Res., 206, 269, 1990. 

KDO synthetase catalyzes the reaction of arabinose 5-phosphate (29 Ara-5-P) and phosphoenol pyruvate (PEP) to form KDO-8-P (30 Scheme 9). KDO synthetase is not commercially available but has been isolated from E. coli and used in the synthesis of KDO-8-P (63% from Ara-5-P, 38 mmol). KDO-8-P is a key intermediate in the synthesis of the lipopolysaccharide region of Gram-negative bacteria (LPS). Inhibitors of LPS biosynthesis are targets for the design of antimicrobial drugs. ... 

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