A 2-deoxy analogue of KDO as the first inhibitor of the enzyme CMP-KDO synthetase

The two KDO analogues 2,6-anhydro-3-deoxy-D-glycero-D-galacto-octonate and 2,6-anhydro-3-deoxy-D-glycero-D-talo-octonate were synthesized and tested as inhibitors of the enzyme CTP:CMP-deoxyoctulosonate cytidylyltransferase (CMP-KDO synthetase) from Gram-negative bacteria. Only compound 4, the 2-deoxy analogue of beta-KDO-pyranose, was found to be an inhibitor with a Ki of 3.9 microM.     

Synthesis of analogues of 3-deoxy-D-manno-octulosonic acid (KDO) as potential inhibitors of CMP-KDO synthetase

A series of derivatives of the 2-deoxy analogue of beta-KDO (2,6-anhydro-3-deoxy-D-glycero-D-talo-octonic acid; ammonium salt, 2) has been synthesised as potential inhibitors of CMP-KDO synthetase, starting from methyl 2,6-anhydro-3-deoxy-4,5:7,8-di-O-isopropylidene-D-glycero-D-talo- octonate and replacing the CO2Me group attached to C-2 variously by CONH2, CONHOH, CH2OH, CH2PO(OH)(O-NH4+), COCH2PO(OH)(O-H3N+pheny), CH2CO2-NH4+, CON-HCH2CO2-NH4+, CONHBn, CONHHexyl, CO2Bn, and CO2Hexyl. Of these derivatives, the hydroxamic acid (CONHOH) was the best inhibitor of CMP-KDO synthetase, but was less potent than 2.     

Characterization of transcription factors and cis-acting elements that regulate human CTP: phosphoethanolamine cytidylyltransferase (Pcyt2)

CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) promoter was isolated from human breast cancer MCF-7 cells and its activity delineated by luciferase reporter assays and gel-shift analysis. The Pcyt2 promoter is driven by a functional CAAT box (-90/-73) and by negative (-385/-255) and positive regulatory elements (-255/-153) in the upstream regions.     

A maize homologue of the bacterial CMP-3-deoxy-D-manno-2-octulosonate (KDO) synthetases. Similar pathways operate in plants and bacteria for the activation of KDO prior to its incorporation into outer cellular envelopes

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.     

A bacteriophage-associated glycanase cleaving beta-pyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO)

A bacteriophage growing on Escherichia coli K13, K20, and K23 strains carries a glycanase that catalyzes the hydrolytic cleavage of the beta-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO) in the respective capsular polysaccharides. The main cleavage product of the K23 polysaccharide has been identified by 1H- and 13C-n.m.r. spectroscopy as beta beta Ribfl----7 beta KDOp2----3-beta Ribfl----7KDO. Cleavage of polysaccharides containing alpha-pyranosidic, or 5-substituted beta-pyranosidic KDO is not catalyzed by the enzyme.     

The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization.     

Metabolic and molecular aspects of ethanolamine phospholipid biosynthesis: the role of CTP:phosphoethanolamine cytidylyltransferase (Pcyt2).

The CDP-ethanolamine branch of the Kennedy pathway is the major route for the formation of ethanolamine-derived phospholipids, including diacyl phosphatidylethanolamine and alkenylacyl phosphatidylethanolamine derivatives, known as plasmalogens. Ethanolamine phospholipids are essential structural components of the cell membranes and play regulatory roles in cell division, cell signaling, activation, autophagy, and phagocytosis. The physiological importance of plasmalogens has not been not fully elucidated, although they are known for their antioxidant properties and deficiencies in a number of inherited peroxisomal disorders. This review highlights important aspects of ethanolamine phospholipid metabolism and reports current molecular information on 1 of the regulatory enzymes in their synthesis, CTP:phosphoethanolamine cytidylyltransferase (Pcyt2). Pcyt2 is encoded by a single, nonredundant gene in animal species that could be alternatively spliced into 2 potential protein products. We describe properties of the mouse and human Pcyt2 genes and their regulatory promoters and provide molecular evidence for the existence of 2 distinct Pcyt2 proteins. The goal is to obtain more insight into Pcyt2 catalytic function and regulation to facilitate a better understanding of the production of ethanolamine phospholipids via the CDP-ethanolamine branch of the Kennedy pathway.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

3-deoxy-d-manno-2-octulosonic acid (KDO) is a component of rhamnogalacturonan II,a pectic polysaccharide in the primary cell walls of plants

3-Deoxy-d-manno-2-octulosonic acid (KDO), a sugar previously presumed to occur only as a glycosyl residue in polysaccharides produced by Gram-negative bacteria, was found to be a component of the cell walls of higher plants. In the form of the disaccharide α-l-Rhap-(1→5)-d-KDO, KDO was released by mild hydrolysis with acid from the purified cell wall polysaccharide rhamnogalacturonan II. KDO was shown to be present in purified cell walls of several plants, including dicots, a monocot, and a gymnosperm. Improved methods for detecting and quantitating KDO residues in polysaccharides were developed during this investigation.     

Synthesis of a trisaccharide of 3-deoxy-D-manno-2-octulopyranosylonic acid (KDO) residues related to the genus-specific lipopolysaccharide epitope of Chlamydia

The disaccharides, O-(sodium 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosylonate)-(2----8)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate, were prepared via glycosylation of methyl (allyl 4,5,7-tri-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosid)onat e with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-D-manno-2-octulopyranosyl bromide)onate under Helferich and Koenigs-Knorr conditions, respectively. Based on g.l.c.-m.s. data of the alpha- and beta-(2----8)-linked disaccharide derivatives, obtained after carbonyl- and carboxyl-group reduction, followed by methylation, the alpha-anomeric configuration was assigned to the terminal KDO-residue in the KDO-region of Chlamydial lipopolysaccharide. The trisaccharide O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate was obtained via block synthesis using an alpha-(2----8)-linked disaccharide bromide derivative as the glycosyl donor. Copolymerization of the allyl glycosides with acrylamide gave water-soluble macromolecular antigens, suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydial LPS.     

Increased phosphatidylcholine production but disrupted glycogen metabolism in fetal type II cells of mice that overexpress CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in the de novo synthesis of phosphatidylcholine (PtdCho). Alveolar type II cells synthesize large quantities of disaturated PtdCho, the surface-active agent of pulmonary surfactant, particularly at late gestation when the lung prepares itself for postnatal air breathing. To clarify the role of CCTalpha in lung surfactant maturation, we overexpressed CCTalpha(1-367) using the surfactant protein-C promoter. Lungs of transgenic mice were analyzed at day 18 of gestation (term = 19 days). Overexpression of CCTalpha(1-367) increased the synthesis and content of PtdCho in fetal type II cells isolated from the transgenic mice. Also, PtdCho content of fetal lung fluid was increased. No changes in surfactant protein content were detected. Interestingly, fetal type II cells of transgenic mice contained more glycogen than control cells. Incorporation studies with [U-(14)C]glucose demonstrated that overexpression of CCTalpha(1-367) in fetal type II cells increased glycogen synthesis without affecting glycogen breakdown. To determine which domain contributes to this glycogen phenotype, two additional transgenes were created overexpressing either CCTalpha(1-239) or CCTalpha(239-367). Glycogen synthesis and content were increased in fetal type II cells expressing CCTalpha(239-367) but not CCTalpha(1-239)(.) We conclude that overexpression of CCTalpha increases surfactant PtdCho synthesis without affecting surfactant protein levels but that it disrupts glycogen metabolism in differentiating type II cells via its regulatory domain.     

Negative cooperativity of substrate binding but not enzyme activity in wild-type and mutant forms of CTP:glycerol-3-phosphate cytidylyltransferase

CTP:glycerol-3-phosphate cytidylyltransferase (GCT) catalyzes the synthesis of CDP-glycerol for teichoic acid biosynthesis in certain Gram-positive bacteria. This enzyme is a model for a cytidylyltransferase family that includes the enzymes that synthesize CDP-choline and CDP-ethanolamine for phosphatidylcholine and phosphatidylethanolamine biosynthesis. We have used quenching of intrinsic tryptophan fluorescence to measure binding affinities of substrates to the GCT from Bacillus subtilis. Binding of either CTP or glycerol-3-phosphate to GCT was biphasic, with two binding constants of about 0.1-0.3 and 20-40 microm for each substrate. The stoichiometry of binding was 2 molecules of substrate/enzyme dimer, so the two binding constants represented distinctly different affinities of the enzyme for the first and second molecule of each substrate. The biphasic nature of binding was observed with the wild-type GCT as well as with several mutants with altered Km or kcat values. This negative cooperativity of binding was also seen when a catalytically defective mutant was saturated with two molecules of CTP and then titrated with glycerol-3-phosphate. Despite the pronounced negative cooperativity of substrate binding, negative cooperativity of enzyme activity was not observed. These data support a mechanism in which catalysis occurs only when the enzyme is fully loaded with 2 molecules of each substrate/enzyme dimer.     

Developmental and metabolic effects of disruption of the mouse CTP:phosphoethanolamine cytidylyltransferase gene (Pcyt2)

The CDP-ethanolamine pathway is responsible for the de novo biosynthesis of ethanolamine phospholipids, where CDP-ethanolamine is coupled with diacylglycerols to form phosphatidylethanolamine. We have disrupted the mouse gene encoding CTP:phosphoethanolamine cytidylyltransferase, Pcyt2, the main regulatory enzyme in this pathway. Intercrossings of Pcyt2(+/-) animals resulted in small litter sizes and unexpected Mendelian frequencies, with no null mice genotyped. The Pcyt2(-/-) embryos die after implantation, prior to embryonic day 8.5. Examination of mRNA expression, protein content, and enzyme activity in Pcyt2(+/-) animals revealed the anticipated 50% decrease due to the gene dosage effect but rather a 20 to 35% decrease. [(14)C]ethanolamine radiolabeling of hepatocytes, liver, heart, and brain corroborated Pcyt2 gene expression and activity data and showed a decreased rate of phosphatidylethanolamine biosynthesis in heterozygotes. Total phospholipid content was maintained in Pcyt2(+/-) tissues; however, this was not due to compensatory increases in the decarboxylation of phosphatidylserine. These results establish the necessity of Pcyt2 for murine development and demonstrate that a single Pcyt2 allele in heterozygotes can maintain phospholipid homeostasis.     

Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid

Substantial evidence indicates that amino acid conjugates of indole-3-acetic acid (IAA) function in auxin homeostasis, yet the plant enzymes involved in their biosynthesis have not been identified. We tested whether several Arabidopsis thaliana enzymes that are related to the auxin-induced soybean (Glycine max) GH3 gene product synthesize IAA-amino acid conjugates. In vitro reactions with six recombinant GH3 enzymes produced IAA conjugates with several amino acids, based on thin layer chromatography. The identity of the Ala, Asp, Phe, and Trp conjugates was verified by gas chromatography-mass spectrometry. Insertional mutations in GH3.1, GH3.2, GH3.5, and GH3.17 resulted in modestly increased sensitivity to IAA in seedling root. Overexpression of GH3.6 in the activation-tagged mutant dfl1-D did not significantly alter IAA level but resulted in 3.2- and 4.5-fold more IAA-Asp than in wild-type seedlings and mature leaves, respectively. In addition to IAA, dfl1-D was less sensitive to indole-3-butyric acid and naphthaleneacetic acid, consistent with the fact that GH3.6 was active on each of these auxins. By contrast, GH3.6 and the other five enzymes tested were inactive on halogenated auxins, and dfl1-D was not resistant to these. This evidence establishes that several GH3 genes encode IAA-amido synthetases, which help to maintain auxin homeostasis by conjugating excess IAA to amino acids.