N-acetylneuraminic Acid Synthetase Research Articles

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC...

Cytidine monophosphate N-acetylneuraminic acid synthetase enhances invasion of human triple-negative breast cancer cells.

BackgroundCancer cells have altered bioenergetics, which contributes to their ability to proliferate, survive in unusual microenvironments, and invade other tissues. Changes in glucose metabolism can have pleomorphic effects on tumor cells.MethodsTo investigate potential mechanisms responsible for the increased malignancy associated with altered glucose metabolism, we used an unbiased nuclear magnetic resonance spectroscopy screening method to identify glucose metabolites differentially produced in a highly malignant human triple-negative breast cancer (TNBC) cell line (BPLER) and a less malignant isogenic TNBC cell line (HMLER).ResultsN-acetylneuraminic acid (Neu5Ac), the predominant sialic acid derivative in mammalian cells, which forms the terminal sugar on mucinous cell surface glycoproteins, was the major glucose metabolite that differed. Neu5Ac was ~7-fold more abundant in BPLER than HMLER. Loss of Neu5Ac by enzymatic removal or siRNA knockdown of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS), which activates cellular sialic acids for glycoprotein conjugation, had no significant effect on cell proliferation, but decreased the ability of BPLER to invade through a basement membrane. Conversely, overexpressing CMAS in HMLER increased invasivity. TNBCs in The Cancer Genome Atlas also had significantly more CMAS copy number variations and higher mRNA expression than non-TNBC, which have a better prognosis. CMAS knockdown in BPLER ex vivo blocked xenograft formation in mice.ConclusionNeu5Ac is selectively highly enriched in aggressive TNBC, and CMAS, the enzyme required for sialylation, may play an important role in TNBC tumor formation and invasivity.

Molecular cloning and bioinformatic analysis of the Streptococcus agalactiae neuA gene isolated from tilapia.

Cytidine monophosphate (CMP) N-acetylneuraminic acid (NeuNAc) synthetase, which is encoded by the neuA gene, can catalyze the activation of sialic acid with CMP, and plays an important role in Streptococcus agalactiae infection pathogenesis. To study the structure and function of the S. agalactiae neuA gene, we isolated it from diseased tilapia, amplified it using polymerase chain reaction (PCR) with specific primers, and cloned it into a pMD19-T vector. The recombinant plasmid was confirmed by PCR and restriction enzyme digestion, and identified by sequencing. Molecular characterization analyses of the neuA nucleotide amino acid sequence were performed using bioinformatic tools and an online server. The results showed that the neuA nucleotide sequence contained a complete coding region, which comprised 1242 bp, encoding 413 amino acids (aa). The aa sequence was highly conserved and contained a Glyco_tranf_GTA_type superfamily and an SGNH_hydrolase superfamily conserved domain, which are related to sialic acid activation catalysis. The NeuA protein possessed many important sites related to post-translational modification, including 28 potential phosphorylation sites and 2 potential N-glycosylation sites, had no signal peptides or transmembrane regions, and was predicted to reside in the cytoplasm. Moreover, the protein had some B-cell epitopes, which suggests its potential in development of a vaccine against S. agalactiae infection. The codon usage frequency of neuA differed greatly in Escherichia coli and Homo sapiens genes, and neuA may be more efficiently expressed in eukaryotes (yeast). S. agalactiae neuA from tilapia maintains high structural homology and sequence identity with CMP-NeuNAc synthetases from other bacteria.

Polynucleotide phosphorylase has an impact on cell biology of Campylobacter jejuni

Polynucleotide phosphorylase (PNPase), encoded by the pnp gene, is known to degrade mRNA, mediating post-transcriptional regulation and may affect cellular functions. The role of PNPase is pleiotropic. As orthologs of the two major ribonucleases (RNase E and RNase II) of Escherichia coli are missing in the Campylobacter jejuni genome, in the current study the focus has been on the C. jejuni ortholog of PNPase. The effect of PNPase mutation on C. jejuni phenotypes and proteome was investigated. The inactivation of the pnp gene reduced significantly the ability of C. jejuni to adhere and to invade Ht-29 cells. Moreover, the pnp mutant strain exhibited a decrease in C. jejuni swimming ability and chick colonization. To explain effects of PNPase on C. jejuni 81-176 phenotype, the proteome of the pnp mutant and parental strains were compared. Overall, little variation in protein production was observed. Despite the predicted role of PNPase in mRNA regulation, the pnp mutation did not induce profound proteomic changes suggesting that other ribonucleases in C. jejuni might ensure this biological function in the absence of PNPase. Nevertheless, synthesis of proteins which are involved in virulence (LuxS, PEB3), motility (N-acetylneuraminic acid synthetase), stress-response (KatA, DnaK, Hsp90), and translation system (EF-Tu, EF-G) were modified in the pnp mutant strain suggesting a more specific role of PNPase in C. jejuni. In conclusion, PNPase deficiency induces limited but important consequences on C. jejuni biology that could explain swimming limitation, chick colonization delay, and the decrease of cell adhesion/invasion ability.

Bacterial CMP-sialic acid synthetases: production, properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell-cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5'-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.

Characterization of a Bifunctional Cytidine 5′-Monophosphate N-acetylneuraminic acid Synthetase Cloned from Streptococcus Agalactiae

Recombinant CMP-sialic acid synthetase, cloned from Streptococcus agalactiae serotype V strain 2603 V/R, is bifunctional having both CMP-sialic acid synthetase and acetylhydrolase (acylesterase) activities. The enzyme is active over a wide pH range with an optimal CMP-sialic acid synthetase activity at pH 9.0 and an optimal acetylhydrolase activity at pH 8.0. A metal cofactor (either Mg(2+) or Mn(2+)) is required for the CMP-sialic acid synthetase activity but is not for acetylhydrolase activity. Both catalytic functions, however, are impaired by high concentrations of Mn(2+).

Expression of human CMP- N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell

Plant cells have no β1,4-galactosylated and sialylated glycan, which plays important roles in biological functions in animal cells. Previously, we generated transgenic tobacco BY2 suspension-cultured cells that produced human β1,4-galactosyltransferase [N.Q. Palacpac, S. Yoshida, H. Sakai, Y. Kimura, K. Fujiyama, T. Yoshida, T. Seki, Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation pattern, Proc. Natl. Acad. Sci. USA 96 (1999) 4692–4697]. In this study, we introduced two critical genes encoding human CMP- N-acetylneuraminic acid synthetase and CMP-sialic acid transporter into tobacco suspension-cultured cell to pave a route for sialic biosynthetic pathway. The recombinant human proteins showed their biological activities. These results show that the plant cell can be a useful bioreactor for the production of mammalian glycoproteins.

Enzymatic Synthesis of Cytidine 5′-Monophospho-N-acetylneuraminic Acid

We have established an efficient method for enzymatic production of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) from inexpensive materials, N-acetylglucosamine (GlcNAc) and cytidine 5'-monophosphate (CMP). The Haemophilus influenzae nanE gene encoding GlcNAc 6-phosphate (GlcNAc 6-P) 2-epimerase and the Campylobacter jejuni neuB1 gene encoding N-acetylneuraminic acid (NeuAc) synthetase, both of whose products are involved in NeuAc biosynthesis, were cloned and co-expressed in Escherichia coli cells. We examined the synthesis of NeuAc from GlcNAc via GlcNAc 6-P, N-acetylmannosamine (ManNAc) 6-P, and ManNAc by the use of E. coli cells producing GlcNAc 6-P 2-epimerase and NeuAc synthetase, in expectation of biological functions of E. coli such as the supply of phosphoenolpyruvate (PEP), which is an essential substrate for NeuAc synthetase, GlcNAc phospholylation by the PEP-dependent phosphotransferase system, and dephospholylation of ManNAc 6-P. Eleven mM NeuAc was synthesized from 50 mM GlcNAc by recombinant E. coli cells with the addition of glucose as an energy source. Next we attempted to synthesize CMP-NeuAc from GlcNAc and CMP using yeast cells, recombinant E. coli cells, and H. influenzae CMP-NeuAc synthetase, and succeeded in efficient production of CMP-NeuAc due to a sufficient supply of PEP and efficient conversion of CMP to cytidine 5'-triphosphate by yeast cells.

Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans.

Previous studies suggested that nontypeable Haemophilus influenzae (NTHI) can form biofilms during human and chinchilla middle ear infections. Microscopic analysis of a 5-day biofilm of NTHI strain 2019 grown in a continuous-flow chamber revealed that the biofilm had a diffuse matrix interlaced with multiple water channels. Our studies showed that biofilm production was significantly decreased when a chemically defined medium lacking N-acetylneuraminic acid (sialic acid) was used. Based on these observations, we examined mutations in seven NTHI strain 2019 genes involved in carbohydrate and lipooligosaccharide biosynthesis. NTHI strain 2019 with mutations in the genes encoding CMP-N-acetylneuraminic acid synthetase (siaB), one of the three NTHI sialyltransferases (siaA), and the undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase homolog (wecA) produced significantly smaller amounts of biofilm. NTHI strain 2019 with mutations in genes encoding phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two other NTHI sialyltransferases (lic3A and lsgB) produced biofilms that were equivalent to or larger than the biofilms produced by the parent strain. The biofilm formed by the NTHI strain 2019pgm mutant was studied with Maackia amurensis fluorescein isothiocyanate (FITC)-conjugated and Sambucus nigra tetramethyl rhodamine isocyanate (TRITC)-conjugated lectins. S. nigra TRITC-conjugated lectin bound to this biofilm, while M. amurensis FITC-conjugated lectin did not. S. nigra TRITC-conjugated lectin binding was inhibited by incubation with alpha2,6-neuraminyllactose and by pretreatment of the biofilm with Vibrio cholerae neuraminidase. Matrix-assisted laser desorption ionization-time of flight mass spectometry analysis of lipooligosaccharides isolated from a biofilm, the planktonic phase, and plate-grown organisms showed that the levels of most sialylated glycoforms were two- to fourfold greater when the lipooligosaccharide was derived from planktonic or biofilm organisms. Our data indicate that NTHI strain 2019 produces a biofilm containing alpha2,6-linked sialic acid and that the sialic acid content of the lipooligosaccharides increases concomitant with the transition of organisms to a biofilm form.

The Crystal Structure of Murine CMP-5- N-acetylneuraminic Acid Synthetase

Sialic acids are activated by CMP-5- N-acetylneuraminic acid synthetase prior to their transfer onto oligo- or polysaccharides. Here, we present the crystal structure of the N-terminal catalytically active domain of the murine 5- N-acetylneuraminic acid synthetase in complex with the reaction product. In contrast to the previously solved structure of 5- N-acetylneuraminic acid synthetase from Neisseria meningitidis and the related CMP–KDO-synthetase of Escherichia coli, the murine enzyme is a tetramer, which was observed with the active sites closed. In this conformation a loop is shifted by 6 Å towards the active site and thus an essential arginine residue can participate in catalysis. Furthermore, a network of intermolecular salt-bridges and hydrogen bonds in the dimer as well as hydrophobic interfaces between two dimers indicate a cooperative behaviour of the enzyme. In addition, a complex regulation of the enzyme activity is proposed that includes phosphorylation and dephosphorylation.

MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides

In a previous report (Young et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5802–5806), we provided a proof-of-principle for fold recognition of proteins using a homobifunctional amine-specific chemical crosslinking reagent in combination with mass spectrometry analysis and homology modeling. In this current work, we propose a systematic nomenclature to describe the types of peptides that are generated after proteolysis of crosslinked proteins, their fragmentation by tandem mass spectrometry, and an automated algorithm for MS/MS spectral assignment called “MS2Assign.” Several examples are provided from crosslinked peptides and proteins including HIV-integrase, cytochrome c, ribonuclease A, myoglobin, cytidine 5-monophosphate N-acetylneuraminic acid synthetase, and the peptide thymopentin. Tandem mass spectra were obtained from various crosslinked peptides using post source decay MALDI-TOF and collision induced dissociation on a quadrupole-TOF instrument, along with their automated interpretation using MS2Assign. A variety of possible outcomes are described and categorized according to the number of modified lysines and/or peptide chains involved, as well as the presence of singly modified (dead-end) lysine residues. In addition, the proteolysis and chromatographic conditions necessary for optimized crosslinked peptide recovery are presented.

Cloning and overexpression of a cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Escherichia coli

The gene for cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC2.7.7.43) was amplified from the total DNA of Escherichia coli 44277 through a primer-directed polymerase chain reaction and cloned into pUC19 between the sites EcoRI and SalI. The sequence analysis showed the cloned DNA was the same as the published neuA except for a codon missing for Asp230 and four base substitutions at the sites 153, 438, 618, and 687. The four base substitutions did not change the amino acid sequence. Based on the preliminary expression experiment and computer-aided analysis of the gene structure, six bases were mutated in order to achieve high-level expression. The gene mutated by PCR was cloned into the expression vector pET15b. Upon the induction of IPTG, the recombinant enzyme was shown to be 26% of the total bacterial proteins, which was 850-fold higher than that of the wild strain. Under the optimal inducing condition, the enzyme yield reached 100 U/l cell culture. Using the partially purified recombinant enzyme, the synthesis of CMP-NeuAc was carried out in 90% yield.

Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide.

N-acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo-oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB-deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild-type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography-mass spectrometry and fast atom bombardment-mass spectrometry analysis of LOS from wild-type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N-acetyl-D-mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non-motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.

Investigation of the kinetic mechanism of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from Haemophilus ducreyi with new insights on rate-limiting steps from product inhibition analysis.

The presence of sialic acid as a component of cell surface lipooligosaccharides or capsular polysaccharides has been shown to be correlated with the virulence of a number of Gram-negative mucosal pathogens, including several Haemophilus and Neisseria spp. As part of our efforts to evaluate the role of sialic acid in the pathobiology of these organisms, we have initiated studies of the enzymes from Haemophilus ducreyi (the infectious agent of chancroid) responsible for the activation and attachment of sialic acid to the lipooligosaccharide. In this report, we describe results of an investigation of the steady-state kinetic mechanism of the activating enzyme, cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. Using a combination of initial velocity, product inhibition, and dead-end inhibition studies, the reaction is shown to be freely reversible and to proceed through an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuAc dissociates last. In addition, a detailed analysis of the kinetic expressions for the observable constants is presented showing how the variation in apparent product inhibition constants (Kii) can be used to predict the rate-limiting step in kcat, which appears to be dissociation of CMP-NeuAc in this enzyme. To our knowledge, this relationship has not been previously recognized.

Covalent modification of Lys19 in the CTP binding site of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase.

Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP.

Identification, expression and tissue distribution of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase activity in the rat.

We report the postnatal developmental profiles of N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-Neu5Ac synthetase) in different rat tissues. This enzyme, which catalyses the activation of NeuAc to CMP-Neu5Ac, was detected in brain, kidney, heart, spleen, liver, stomach, intestine, lung, thymus, prostate and urinary bladder but not in skeletal muscle. Comparative analysis of the different specific activity profiles obtained shows that the expression of CMP Neu5Ac synthetase is tissue-dependent and does not seem to be embryologically determined. Changes in the level of sialylation during development were also found to be intimately related to variations in the expression of this enzyme, at least in brain, heart, kidney, stomach, intestine and lung.

Purification, Cloning, and Expression of a Cytidine 5′-Monophosphate N-Acetylneuraminic Acid Synthetase from Haemophilus ducreyi

An N-acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CMP-NeuAc synthetase) was isolated from a Haemophilus ducreyi strain 35000 cell lysate and partially characterized. The enzyme catalyzes the reaction of CTP and NeuAc to form CMP-NeuAc, which is the nucleotide sugar donor used by sialyltransferases. Previous studies have shown that the outer membrane lipooligosaccharides of H. ducreyi contain terminal sialic acid attached to N-acetyllactosamine and that this modification is likely important to its pathogenesis. Therefore, to investigate the role of sialic acid in H. ducreyi pathogenesis, the gene encoding the CMP-NeuAc synthetase was cloned using degenerate oligonucleotide probes derived from NH2-terminal sequence data, and the nucleotide sequence was determined. The derived amino acid sequence of the CMP-NeuAc synthetase gene has homology to other CMP-NeuAc synthetases and to a lesser extent to CMP-2-keto-3-deoxy-D-manno-octulosonic acid synthetases. The gene was cloned into a T7 expression vector, the protein expressed in Escherichia coli, and purified to apparent homogeneity by anion exchange, Green 19 dye, and hydrophobic interaction chromatography. The final step yielded 20 mg of pure protein/liter of culture. The protein has a predicted molecular mass of 25440.6 Da, which was confirmed by electrospray mass spectrometry (Mexpt = 25439.9 +/- 1.4 Da). The enzyme appears to exist as a dimer by size exclusion chromatography. In contrast to other bacterial CMP-NeuAc synthetases, the H. ducreyi enzyme exhibited a different substrate specificity, being capable of also using N-glycolylneuraminic acid as a substrate.

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP- N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.

Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.

Regulation of colominic acid biosynthesis by temperature: Role of cytidine 5′-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5′-monophospho- N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.