The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐<i>manno</i>‐octonic acid synthetase from <i>Escherichia coli</i>

Jelakovic, S.; Jann, Klaus; Schulz, Georg E.

doi:10.1016/0014-5793(96)00724-7

Cited by 31 publications

(21 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In agreement with this work, amino acid sequence analysis, site-directed mutagenesis, and chemical modification studies indicate residues 10 -22 (phosphate binding loop or P-loop) following strand ␤1 are integral components of the mononucleotide-binding pocket (33,40). Likewise, in the CMPKDOS structure (38), residues of the P-loop were observed to bind a heavy atom derivative (IrCl 6 3Ϫ ), and the authors speculated that these residues play a role in phosphate binding.…”

Section: Resultssupporting

confidence: 57%

“…The similarities between the N terminus of CMPNeuAc synthetase and Rossman fold containing proteins are restricted to the order and connectivity of secondary structures, because CMP-NeuAc synthetase and related enzymes have unique mononucleotide-binding pockets. The CMP-NeuAc synthetase ␣/␤ domain fold is similar to that reported for cytosine-5Ј-monophosphate-2-keto-3-deoxy-manno-octonate synthetase (CMPKDOS (38)), a homologous, dimeric enzyme that synthesizes a related, activated sugar compound, cytosine-5Ј-monophosphate 2-keto-3-deoxy-manno-octonate (CMP-KDO). These two enzymes are similar in length but share limited amino acid sequence identity (18%, Fig.…”

Section: Resultssupporting

confidence: 48%

See 1 more Smart Citation

Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP

Mosimann

Gilbert²,

Dombroswki

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The x-ray crystallographic structure of selenomethionyl cytosine-5-monophosphate-acylneuraminate synthetase (CMP-NeuAc synthetase) from Neisseria meningitidis has been determined at 2.0-Å resolution using multiple-wavelength anomalous dispersion phasing, and a second structure, in the presence of the substrate analogue CDP, has been determined at 2.2-Å resolution by molecular replacement. This work identifies the active site residues for this class of enzyme for the first time. The detailed interactions between the enzyme and CDP within the mononucleotide-binding pocket are directly observed, and the acylneuraminate-binding pocket has also been identified. A model of acylneuraminate bound to CMP-NeuAc synthetase has been constructed and provides a structural basis for understanding the mechanism of production of "activated" sialic acids. Sialic acids are key saccharide components on the surface of mammalian cells and can be virulence factors in a variety of bacterial species (e.g. Neisseria, Haemophilus, group B streptococci, etc.). As such, the identification of the bacterial CMP-NeuAc synthetase active site can serve as a starting point for rational drug design strategies.Cytosine-5Ј-monophosphate-acylneuraminate synthetase (cytosine-5Ј-monophosphate-N-acetyl neuraminic acid synthetase, CMP-NeuAc synthetase) 1 catalyzes the penultimate step in the addition of sialic acids to the oligosaccharide component of glycoconjugates and is a required component of sialylation pathways. CMP-NeuAc synthetase-deficient mutants do not express sialylated glycoconjugates and can be complemented with functional CMP-NeuAc synthetase in both mammalian (1) and bacterial systems (2). Sialic acids participate in a myriad of signaling, recognition, and cell-cell adhesion phenomena in mammalian cells (3) and are overexpressed in some highly malignant tumors (4). Genetic disorders that lead to altered physiological levels of sialic acids have many consequences in humans and can be fatal (5). In bacterial systems, sialic acids are less common and frequently have roles as virulence factors (6). In Neisseria gonorrhoeae, sialylated glycoconjugates protect the organism from phagocytosis and increase serum resistance (7). In Haemophilus ducreyi the presence of 2-keto-3-deoxy-manno-octulosonate-containing glycoconjugates correlate with the organism's pathogenicity (8). Likewise, the sialylated capsule of Neisseria meningitidis, Escherichia coli K1, and group B streptococci are virulence factors (9-11). It has been suggested that bacterial species produce sialylated glycoconjugates to mimic the host and escape detection by the immune system (12). Clearly, the mechanism of production of sialic acid-containing glycoconjugates is of clinical interest. Although bacterial and eucaryotic CMPNeuAc synthetase enzymes share many catalytic properties, several important differences have been reported, including substrate specificity, tertiary structure, inhibitor sensitivity, and cellular localization (13). As a result, bacterial CMP-NeuAc synthetase en...

show abstract

Section: Resultssupporting

confidence: 57%

Section: Resultssupporting

confidence: 48%

Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP

Mosimann

Gilbert²,

Dombroswki

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…We therefore conclude that AA-LCKS forms dimers, like the related CKS enzymes of E. coli and H. influenzae [20], [21], [23] and the homologous CNS of Neiseria meningitidis [17]. AA-LCKS was found to be active in the temperature range between 30°C and 95°C.…”

Section: Resultsmentioning

confidence: 74%

Evidence for a Two-Metal-Ion Mechanism in the Cytidyltransferase KdsB, an Enzyme Involved in Lipopolysaccharide Biosynthesis

Schmidt

Mesters

et al. 2011

PLoS ONE

View full text Add to dashboard Cite

Lipopolysaccharide (LPS) is located on the surface of Gram-negative bacteria and is responsible for maintaining outer membrane stability, which is a prerequisite for cell survival. Furthermore, it represents an important barrier against hostile environmental factors such as antimicrobial peptides and the complement cascade during Gram-negative infections. The sugar 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an integral part of LPS and plays a key role in LPS functionality. Prior to its incorporation into the LPS molecule, Kdo has to be activated by the CMP-Kdo synthetase (CKS). Based on the presence of a single Mg2+ ion in the active site, detailed models of the reaction mechanism of CKS have been developed previously. Recently, a two-metal-ion hypothesis suggested the involvement of two Mg2+ ions in Kdo activation. To further investigate the mechanistic aspects of Kdo activation, we kinetically characterized the CKS from the hyperthermophilic organism Aquifex aeolicus. In addition, we determined the crystal structure of this enzyme at a resolution of 2.10 Å and provide evidence that two Mg2+ ions are part of the active site of the enzyme.

show abstract

“…This and their essential role in bacterial viability make these enzymes an attractive target for the design of very selective antimicrobial agents (10,11). However, the sugar KDO is not restricted to Gram-negative bacteria.…”

mentioning

confidence: 99%

A Maize Homologue of the Bacterial CMP-3-Deoxy-d-manno-2-octulosonate (KDO) Synthetases

Royo

Gómez

Hueros

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The eight-carbon acid sugar 3-deoxy-D-manno-2-octulosonate (KDO) is an essential component of Gram-negative bacterial cell walls and capsular polysaccharides. KDO is incorporated into these polymers as CMP-KDO, which is produced in an unusual activation step catalyzed by the enzyme CMP-KDO synthetase. CMP-KDO synthetase activity has traditionally been considered exclusive to Gram-negative bacteria. CMP-KDO synthetase inhibitors attract great interest owing to their potential as selective bactericides. The sugar KDO is also a component of the rhamnogalacturonan II pectin fraction of the primary cell walls of most higher plants and of the cell wall polysaccharides of some green algae. However, the metabolic pathway leading to its incorporation into the plant cell wall is unknown. This paper describes the isolation and characterization of a maize gene, which codes for a protein very similar in sequence and activity to prokaryotic CMP-KDO synthetases. Remarkably, the maize gene can complement a CMP-KDO synthetase (kdsB) Salmonella typhimurium mutant defective in cell wall synthesis. ZmCKS activity is novel in eukaryotes. The evolutionary origin of ZmCKS is discussed in relation to the high degree of conservation between the plant and bacterial genes and its atypical codon usage in maize. The acid sugar 3-deoxy-D-manno-octulosonate (KDO)1 is an important component of the lipopolysaccharides found on the outer surface of Gram-negative bacteria (1, 2), where it serves as the link between lipid A and core oligosaccharides. KDO is also found in some capsular polysaccharides of the Gram-negative bacteria (3-5).The incorporation of KDO into lipopolysaccharides is catalyzed by membrane-bound KDO-transferase that requires a nucleotide derivative of the molecule, CMP-KDO (1). The synthesis of this activated form of KDO is catalyzed by the enzyme CMP-KDO synthetase (E.C. 2.7.7.38) in a reaction that takes place in the bacterial cytoplasm.Activation of a sugar by its coupling to monophosphonucleosides rather than to diphosphonucleosides is very unusual. The only other known sugar activation reaction with a similar mechanism is the formation of CMP-N-acetylneuraminic acid (6).CMP-KDO synthetase is an essential gene product for the growth of most Gram-negative bacterial cells. Temperaturesensitive mutations in CMP-KDO synthetase (kdsB gene) are lethal at the nonpermissive temperature because the incorporation of KDO into lipid A precursors is blocked. These lipid A precursors then accumulate, inhibit cell growth, and ultimately cause death (7,8). A second, nonessential CMP-KDO synthetase gene involved in the synthesis of capsular polysaccharides has also been described in Escherichia coli (kpsU gene; Ref. 9).CMP-KDO synthetases are traditionally considered unique to Gram-negative bacteria. This and their essential role in bacterial viability make these enzymes an attractive target for the design of very selective antimicrobial agents (10, 11). However, the sugar KDO is not restricted to Gram-negative bacteria. It is also a compon...

show abstract

The three‐dimensional structure of capsule‐specific CMP: 2‐keto‐3‐deoxy‐manno‐octonic acid synthetase from Escherichia coli

Cited by 31 publications

References 36 publications

Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP

Structure of a Sialic Acid-activating Synthetase, CMP-acylneuraminate Synthetase in the Presence and Absence of CDP

Evidence for a Two-Metal-Ion Mechanism in the Cytidyltransferase KdsB, an Enzyme Involved in Lipopolysaccharide Biosynthesis

A Maize Homologue of the Bacterial CMP-3-Deoxy-d-manno-2-octulosonate (KDO) Synthetases

Contact Info

Product

Resources

About