Effect of Altered Lipid A Synthesis on the Synthesis of Major Proteins of the Salmonella typhimurium Outer Membrane

Rick, Paul D.; Neumeyer, B.; Young, Debra A.

doi:10.1093/clinids/6.4.455

Cited by 7 publications

(9 citation statements)

References 20 publications

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“…Interestingly, the E. coli DAH7P synthase can synthesize KDO8P from A5P and PEP at modest rates (36), whereas KDO8P synthase cannot catalyze the synthesis of DAH7P from E4P and PEP. 5 Clearly, the active site of DAH7P synthase must be able to accommodate the longer A5P. In contrast, the binding of E4P and PEP in the active site of KDO8P synthase would leave a gap of approximately 3.5 Å between C1 E4P and C3 PEP , decreasing the probability of forming a bond between these two atoms.…”

Section: Resultsmentioning

confidence: 99%

“…The H268G mutant of DAH7P synthase is also completely inactive. 5 Thus, it is tempting to speculate that this residue might be intimately involved in the catalytic mechanism. For example, if KDO8P synthesis occurs according to the pathway described by Mechanism I, the stereochemistry of the reaction will be determined by the initial attack of a water molecule at C2…”

Section: Resultsmentioning

confidence: 99%

“…Dephosphorylation of KDO8P to KDO and synthesis of CMP-KDO (from CTP and KDO) occur prior to insertion of the sugar into LPS (2). Strains of Salmonella have been isolated with mutations in KDO8P synthase that confer temperature-sensitive growth (4,5). Such strains fail to synthesize KDO at the nonpermissive temperature, which leads to the inhibition of LPS biosynthesis and, as a consequence, to the arrest of cell growth.…”

Section: -Deoxy-d-manno-octulosonate (Kdo)mentioning

confidence: 99%

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Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

Radaev¹,

Dastidar²,

Patel³

et al. 2000

Journal of Biological Chemistry

137

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3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate. KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria. The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 Å (R-factor, 19.9%; R-free, 23.9%). KDO8P synthase is a homotetramer in which each monomer has the fold of a (␤/␣) 8 barrel. On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 Å apart such that KDO8P synthesis would proceed via a linear intermediate. A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase. In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase. This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. 3-Deoxy-D-manno-octulosonate (KDO)1 is an 8-carbon sugar present in the lipopolysaccharide (LPS) of all Gram-negative bacteria (1). KDO provides a link between lipid A, the membrane embedded moiety of LPS, and the O-antigen, an elongated polysaccharide chain that protrudes from the bacterial outer membrane into the surrounding environment and determines the antigenic specificity of the cell. Although the composition of the O-antigen varies between species and also between strains, the inner core region containing KDO is fairly constant among all Gram-negative bacteria (2).3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8P synthase, EC 4.1.2.16) plays a key role in the biosynthesis of KDO. This enzyme catalyzes the aldol-type condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P (precursor to KDO) and inorganic phosphate ( Fig. 1) (3). Dephosphorylation of KDO8P to KDO and synthesis of CMP-KDO (from CTP and KDO) occur prior to insertion of the sugar into LPS (2). Strains of Salmonella have been isolated with mutations in KDO8P synthase that confer temperature-sensitive growth (4, 5). Such strains fail to synthesize KDO at the nonpermissive temperature, which leads to the inhibition of LPS biosynthesis and, as a consequence, to the arrest of cell growth. These studies indicate that KDO8P synthase provides an essential function for bacterial homeostasis.Earlier studies have determined that the reaction of KDO8P synthesis is a sequential process in which the binding of PEP precedes the binding of A5P and the release of inorganic phosphate precedes the release of KDO8P (6). The condensation step of the reaction is stereospecific, involving the additio...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: -Deoxy-d-manno-octulosonate (Kdo)mentioning

confidence: 99%

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Structure and Mechanism of 3-Deoxy-d-manno-octulosonate 8-Phosphate Synthase

Radaev¹,

Dastidar²,

Patel³

et al. 2000

Journal of Biological Chemistry

137

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“…KDO8P is the precursor to 3-deoxy-D-manno-octulosonic acid (KDO), an unusual octulose found in the inner core of lipopolysaccharide. KDO assumes an important role in the overall assembly of lipopolysaccharide, and its production and utilization have proven central to cellular viability and homeostasis (22)(23)(24)(25). In the reaction catalyzed by DAHPS, D-erythrose 4-phosphate (E4P) serves as the aldose cosubstrate yielding the heptulose, DAHP.…”

mentioning

confidence: 99%

A Metal Bridge between Two Enzyme Families

Duewel

Woodard

2000

Journal of Biological Chemistry

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The enzymes 3-deoxy-D-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and D-arabinose 5-phosphate or D-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. , and Zn 2؉ and is reversibly inhibited by higher concentrations (>1 mM) of certain metals. Analysis of several metal forms of the enzyme by plasma mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.Over the past several years, our understanding of a unique class of enzymes responsible for the incorporation of the 3-carbon skeleton of phosphoenolpyruvate (PEP) 1 into pivotal biosynthetic intermediates has dramatically increased. Common to this particular enzyme class, which can be divided into two broad groups, is cleavage of the C-O bond of PEP concurrent with catalysis, as opposed to the more conventional cleavage of the P-O bond (1-7). Members of the first group, 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvate transferase, promote transfer of the intact carboxyvinyl portion of PEP to a cosubstrate alcohol with the formation of an enolic ether linkage to the C-2 of PEP. The mechanisms of enolpyruvylshikimate-3-phosphate synthase (8 -12) and UDP-N-acetylglucosamine enolpyruvate transferase (13-15) have been thoroughly characterized.The second group of enzymes associated with C-O bond cleavage reactions presently includes 3-deoxy-D-manno-octulosonic acid-8-phosphate (KDO8P) synthase (KDO8PS) (16, 17) and 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (DAHPS) (18,19). Both enzymes catalyze the condensation of PEP with a phosphorylated monosaccharide via coupling of the C-3 of PEP to the C-1 of an aldose cosubstrate to produce a 3-deoxy-2-keto sugar acid three carbons longer. KDO8PS occupies an essential position in the biosynthesis of lipopolysaccharide in Gram-negative microorganisms (20, 21), using D-arabinose 5-phosphate (A5P) in its condensation reaction to yield KDO8P and inorganic phosphate. KDO8P is the precursor to 3-deoxy-D-manno-octulosonic acid (KDO), an unusual octulose found in the inner core of lipopolysaccharide. KDO assumes an important role in the overall assembly of lipopolysaccharide, and its production and utilization have proven central to cellular viability and homeostasis (22)(23)(24)(25). In the reaction catalyzed by DAHPS, D-erythrose 4-phosphate (E4P) serves as the...

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“…Alternatively, the extracted products were treated directly with mild acid, and the radioactive oligosaccharides released from the glycolipids were analyzed as described below. (8) and analyzed by fluorography with En3Hance (20).…”

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confidence: 99%