Kdo transferase

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. 

The final enzyme in the pathway, CMP-KDO transferase, the enzyme that catalyzes the transfer of KDO from CMP-KDO to the Lipid A acceptor has been studied in Mary Jane Osborn s laboratory. We have not further purified this enzyme nor studied its properties in detail. The in vitro reaction described by Munson et al. (15) utilized a detergent solubilized enzyme preparation to catalyze the transfer of two KDO residues to the Lipid A precursor. 

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... 

KDO-transferase KdtA (multifunctional) Chlamydia pneumoniae Z31593 41... 

WiDTE, K. A. Kaltashov, I. A. Cotter, R. 1. Raetz, C. R. H. A mono-functional 3-deoxy-D-manno-octulosonic acid (Kdo) transferase and a Kdo kinase in extracts of Haemophilus influenzae. Journal of Biological Chemistry 1997, 272, 16555—16563. 

The Kdo transferase from E coH has been cloned, overexpressed and purified [35]. The purified protein displays at least 1000-fold preference for substrates which contain a 4 phosphate group, indicating the importance of the 4 kinase in lipid A biosynthesis [35]. 

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... 

A -acetyl-D-neuraminic acid is biosynthesized from A -acetyl-D-mannosamine and phosphoenol pyruvate, catalyzed by N-acetyl-D-neuraminic acid synthase. The first step involves the addition of an electron pair from the double bond of the phosphoenol pyruvate to the aldehyde group to give an aldol-type condensation (see Fig. 10.8A). The product is the nine-carbon sugar acid, A -acetyl-D-neuraminic acid [23]. In some instances the enzyme requires A-acetyl-D-mannosamine-6-phosphate as the substrate and forms A-acetyl-D-neuraminic acid-9-phosphate. Various hydroxyl groups on C-4, -7, -8, and -9 can be acetylated by specific acetyl transferases using acetyl CoAas the donor. KDO (2-keto-3-deoxy-D-mannooctulosonic acid) is biosynthesized by a very similar condensation between D-arabinose-5-phosphate and pyruvic acid, catalyzed by KDO synthase (see Fig. 10.8B) [24]. 

Lipid A, which anchors lipopolysaccharide in the membrane, is made first. Hydroxy acids are added first to the disaccharide, followed by KDO and then saturated fatty acids. The hydroxy fatty acids come from acyl-CoA substrates whereas CMP-KDO is the source of the second addition units. After the addition of saturated fatty acids, sugars are added from nucleotide diphosphate derivatives. Various deficient mutants which lack either glucosyl- or galactosyl-transferase have been isolated. These reactions build one half of the molecule. Another lipid, phosphatidylethanolamine, has been suggested to be intimately involved in the binding of the transferase enzymes to the lipopolysaccharide acceptor. 

---