Purification and Characterization of a Specific 3-Deoxy- d - manno -Octulosonate 8-Phosphate Phosphatase from Escherichia coli B

Abstract

A phosphatase specific for the hydrolysis of 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate was purified approximately 400-fold from crude extracts of Escherichia coli B. The hydrolysis of KDO-8-phosphate to KDO and inorganic phosphate in crude extracts of E. coli B, grown in phosphate-containing minimal medium, could be accounted for by the enzymatic activity of this specific phosphatase. No other sugar phosphate tested was an alternate substrate or inhibitor of the purified enzyme. KDO-8-phosphate phosphatase was stimulated threeto fourfold by the addition of 1.0 mM Co' or Mg2" and to a lesser extent by 1.0 mM Ba2 , Zn2+, and Mn2. The activity was inhibited by the addition of 1.0 mM ethylenediaminetetraacetic acid, Cu2+, Ca2+, Cd2+, Hg2+, and chloride ions (50% at 0.1 M). The pH optimum was determined to be 5.5 to 6.5 in both tris(hydroxymethyl)aminomethane-acetate and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer. This specific phosphatase had an isoelectric point of 4.7 to 4.8 and a molecular weight of 80,000 ± 6,000 as determined by molecular sieving and Ferguson analysis. The enzyme appeared to be composed of two identical subunits of 40,000 to 43,000 molecular weight. The apparent Km for KDO-8-phosphate was determined to be 5.8 ± 0.9 x 10' M in the presence of 1.0 mM Co +, 9.1 ± 1 x 10' M in the presence of 1.0 mM Mg2+, and 1.0 ± 0.2 x 10-4 M in the absence of added Co2+ or Mg2+. 3-Deoxy-D-manno-octulosonic acid (KDO) is a unique sugar that is an integral part of the lipopolysaccharide region of most gram-negative bacteria. This eight-carbon keto sugar is the direct link between lipid A and the growing polysaccharide chain (10, 23, 25, 26). Osborn and co-workers (16, 20, 24, 26) have isolated temperature-sensitive mutants defective in KDO biosynthesis and demonstrated the involvement of KDO in the maturation of lipid A. The biosynthesis and utilization of KDO is known to involve at least five sequential reactions: D-ribulose 5-phosphate D-arabinose 5-phosphate D-arabinose 5-phosphate + phosphoenolpyru-(2) vate (PEP)-KDO-8-phosphate + Pi KDO-8-phosphate-* KDO + Pi

Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase.

“…The specific activity of CMP-KDO synthetase in crude extracts of E. coli B grown on glucose CMP-KDO SYNTHETASE 1279 miniimal medium is in the range required for lipopolysaccharide synthesis in vivo (2 nmol min-' mg of protein-' [data not shown]), and would suggest that the formation of CMP-KDO may be the rate-limiting step in lipopolysaccharide biosynthesis. The specific activities in crude extracts of E. coli B of D-arabinose-5-phosphate isomerase, KDO-8-phosphate synthase, and KDO-8-phosphate phosphatase are at least 15-fold greater than that of CMP-KDO synthetase (17,18). Also of interest is the substrate specificity of CMP-KDO synthetase.…”

“…The biosynthesis and metabolism of KDO involve at least four sequential reactions after the formation of D-arabinose-5-phosphate (10, These four reactions are catalyzed by KDO-8phosphate synthase (9,17), KDO-8-phosphate phosphatase (3,18), CMP-KDO synthetase (5), and KDO transferase(s) (13), respectively. In previous publications, we have reported the purification and charact ization of KDO-8-phosphate synthase (17) and a specific KDO-8-phosphate phosphatase (18). In this paper, we describe the purification and characterization of CMP-KDO synthetase from Escherichia coli B.…”

“…Protein was measured by the method of Lowry et al (11), using bovine serum albumin as the standard. Pi was determined by the method of Ames (1), using KH2PO4 as the standard as described previously (17,18). The formation of CMP-KDO was determined by a modification of the thiobarbituric acid assay utilized by Kean and Roseman (8) for the determination of CMP-sialic acid and by Ghalambor and Heath (5) for the determination of CMP-KDO.…”

Invited review: Lipopolysaccharide biosynthesis: which steps do bacteria need to survive?

“…The gene encoding this enzyme is still unknown; however, the enzyme was purified to homogeneity and its biochemical properties are well characterized. 78 To incorporate Kdo into LPS it has to be activated to the nucleotide sugar CMP-Kdo. This reaction is catalyzed by CMP-Kdo synthetase, KdsB.…”

Molecular cloning of the structural gene coding for CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase from Escherichia coli K-12

“…KDO is synthesized in vivo by two enzymatic steps as follows: (i) D-arabinose- 5-phosphate + phosphoenolpyruvate -* KDO-8-phosphate + Pi, and (ii) KDO-8-phosphate -* KDO + Pi. Reactions (i) and (ii) are catalyzed by KDO-8-phosphate synthetase (10,14) and KDO-8-phosphate phosphatase (15), respectively (18). Before incorporation to lipid A precursor, KDO is activated to CMP-KDO by reaction (iii), CTP + KDO -+ CMP-KDO + PP, (16), and subsequently donated to lipid A precursor in reaction (iv), CMP-KDO + lipid A precursor--CMP + lipid A precursor-KDO (11).…”