Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase

Quinolinate synthetase catalyzes the second step of the de novo biosynthetic pathway of pyridine nucleotide formation. In particular, quinolinate synthetase is involved in the condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinic acid. To study the mechanism of action, the specificity of the enzyme and the interaction with l-aspartate oxidase, the other component of the so-called "quinolinate synthetase complex," the cloning, the overexpression, and the purification to homogeneity of Escherichia coli quinolinate synthetase were undertaken. The results are presented in this paper. Since the overexpression of the enzyme resulted in the formation of inclusion bodies, a procedure of renaturation and refolding had to be set up. The overexpression and purification procedure reported in this paper allowed the isolation of 12 mg of electrophoretically homogeneous quinolinate synthetase from 1 liter of E. coli culture. A new, continuous, method for the evaluation of quinolinate synthetase activity was also devised and is presented. Finally, our data definitely exclude the possibility that other enzymes are involved in the biosynthesis of quinolinic acid in E. coli, since it is possible to synthesize quinolinic acid from l-aspartate, dihydroxyacetone phosphate, and O(2) by using only nadA and nadB gene overexpressed products.     

Quinolinate synthetase, an iron-sulfur enzyme in NAD biosynthesis

Nicotinamide adenine dinucleotide (NAD) plays a crucial role as a cofactor in numerous essential redox biological reactions. NAD derives from quinolinic acid which is synthesized in Escherichia coli from L-aspartate and dihydroxyacetone phosphate (DHAP) as the result of the concerted action of two enzymes, L-aspartate oxidase (NadB) and quinolinate synthetase (NadA). We report here the characterization of NadA protein from E. coli. When anaerobically purified, the isolated soluble protein contains 3-3.5 iron and 3-3.5 sulfide/polypeptide chain. M?ssbauer spectra of the 57Fe-protein revealed that the majority of the iron is in the form of a (4Fe-4S)2+ cluster. An enzymatic assay for quinolinate synthetase activity was set up and allowed to demonstrate that the cluster is absolutely required for NadA activity. Exposure to air leads to degradation of the cluster and inactivate enzyme.     

Purification of PII and PII-UMP and In Vitro Studies of Regulation of Glutamine Synthetase in Rhodospirillum rubrum

The P(II) protein from Rhodospirillum rubrum was fused with a histidine tag, overexpressed in Escherichia coli, and purified by Ni(2+)-chelating chromatography. The uridylylated form of the P(II) protein could be generated in E. coli. The effects on the regulation of glutamine synthetase by P(II), P(II)-UMP, glutamine, and alpha-ketoglutarate were studied in extracts from R. rubrum grown under different conditions. P(II) and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by alpha-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the E. coli system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of Superoxide Dismutase by Molecular Oxygen

Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O(2) had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O(2) caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O(2) rather than to pressure, since 20 atm of N(2) was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N(2)-grown cells of S. faecalis were exposed to 20 atm of O(2) at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O(2) during growth. S. faecalis, which had been grown under 100% O(2) and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O(2) than were cells which had been grown under N(2). E. coli grown under N(2) contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O(2). The E. coli which had been grown under N(2) was as resistant to the deleterious effects of 50 atm of O(2) as was S. faecalis which had been grown under 1 atm of O(2). These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.     

Fasciola hepatica: heterologous expression and functional characterization of a thioredoxin peroxidase

A Fasciola hepatica cDNA clone of 779 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 582 bp which encoded a 194-amino-acid-residue polypeptide (M(r) 21,723 Da) showing a high degree of homology to thioredoxin peroxidases. This putative antioxidant protein gene was expressed in Escherichia coli as a GST fusion protein. The recombinant fusion protein showed in vitro antioxidant properties and protected rabbit muscle enolase and E. coli glutamine synthetase from inactivation by nonenzymatic Fe(3+)/O(2)/DTT or Fe(3+)/O(2)/ascorbate metal-catalyzed oxidation systems.     

Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.     

Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase.

The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain. Growth of these strains on a gluconate minimal medium was also impaired under these conditions. The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation. The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E. coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1. The dehydratase was much less sensitive to O2 or H2O2 than to O2-. The virtual substrate, alpha-glycerophosphate, provided partial protection. Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme. Fluoride ions stabilized the dehydratase and blocked the effect of oxidants. The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.     

Oxidation of Neurospora crassa glutamine synthetase.

The glutamine synthetase of Neurospora crassa, either purified or in cell extracts, was inactivated by ascorbate plus FeCl3 and by H2O2 plus FeSO4. The inactivation reaction was oxygen dependent, inhibited by MnCl2 and EDTA, and stimulated in cell extracts by sodium azide. This inactivation could also be brought about by adding NADPH to the cell extract. The alpha and beta polypeptides of the active glutamine synthetase were modified by these inactivating reactions, giving rise to two novel acidic polypeptides. These modifications were observed with the purified enzyme, with cell extracts, and under in vivo conditions in which glutamine synthetase is degraded. The modified glutamine synthetase was more susceptible to endogenous phenylmethylsulfonyl fluoride-insensitive proteolytic activity, which was inhibited by MnCl2 and stimulated by EDTA. The possible physiological relevance of enzyme oxidation is discussed.     

Oxygen regulation of ribulose 1,5-bisphosphate carboxylase activity in Rhodospirillum rubrum.

The carboxylase activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O) decreased when an anaerobic culture of Rhodospirillum rubrum was exposed to atmospheric levels of oxygen. From 70 to 80% of the activity was lost within 12 to 24 h. Inactivation was apparent when the enzyme was assayed in situ (in whole cells) and when activity was measured in dialyzed crude extracts. The quantity of enzyme protein, as estimated from sodium dodecyl sulfate-polyacrylamide gels or as quantified immunologically, did not decrease within 24 h of exposure to air. Following extended exposure to aerobic conditions (48 to 72 h), degradation of enzyme occurred. These results indicate that the inactivation of RuBPC/O in R. rubrum may be due to an alteration or modification of the preformed enzyme, followed by eventual degradation of the inactive enzyme. When shifted back to anaerobic conditions (under an argon atmosphere), the RuBPC/O activity increased rapidly. This increase appeared to be due to de novo synthesis of enzyme. The increase in activity was not observed when the culture was maintained in the dark or in the absence of a suitable carbon source. Thus, the oxygen-mediated inactivation of RuBPC/O appeared to be due to some form of irreversible modification. The cloned R. rubrum RuBPC/O gene, expressed in Escherichia coli, yielded functional enzyme that was not affected by oxygen, indicating that inactivation in R. rubrum is mediated by a gene product(s) not found in E. coli.     

Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum.

We have isolated the Bradyrhizobium japonicum gene encoding glutamine synthetase I (glnA) from a phage lambda library by using a fragment of the Escherichia coli glnA gene as a hybridization probe. The rhizobial glnA gene has homology to the E. coli glnA gene throughout the entire length of the gene and can complement an E. coli glnA mutant when borne on an expression plasmid in the proper orientation to be transcribed from the E. coli lac promoter. High levels of glutamine synthetase activity can be detected in cell-free extracts of the complemented E. coli. The enzyme encoded by the rhizobial gene was identified as glutamine synthetase I on the basis of its sedimentation properties and resistance to heat inactivation. DNA sequence analysis predicts a high level of amino acid sequence homology among the amino termini of B. japonicum, E. coli, and Anabaena sp. strain 7120 glutamine synthetases. S1 nuclease protection mapping indicates that the rhizobial gene is transcribed from a single promoter 131 +/- 2 base pairs upstream from the initiation codon. This glnA promoter is active when B. japonicum is grown both symbiotically and in culture with a variety of nitrogen and carbon sources. There is no detectable sequence homology between the constitutively expressed glnA promoter and the differentially regulated nif promoters of the same B. japonicum strain.     

Inactivation of glutamine synthetase by a purified rabbit liver microsomal cytochrome P-450 system

Several mixed-function oxidation systems catalyze inactivation of Escherichia coli glutamine synthetase and other key metabolic enzymes. In the presence of NADPH and molecular oxygen, highly purified preparations of cytochrome P-450 reductase and cytochrome P-450 (isozyme 2) from rabbit liver microsomes catalyze enzyme inactivation. The inactivation reaction is stimulated by Fe(III) or Cu(II) and is inhibited by catalase, Mn(II), Zn(II), histidine, and the metal chelators o-phenanthroline and EDTA. The inactivation of glutamine synthetase is highly specific and involves the oxidative modification of a histidine in each glutamine synthetase subunit and the generation of a carbonyl derivative of the protein which forms a stable hydrazone when treated with 2,4-dinitrophenylhydrazine. We have proposed that the mixed-function oxidation system (the cytochrome P-450 system) produces Fe(II) and H2O2 which react at the metal binding site on the glutamine synthetase to generate an activated oxygen species which oxidizes a nearby susceptible histidine. This thesis is supported by the fact that (a) Mn(II) and Zn(II) inhibit inactivation and also interfere with the reduction of Fe(III) to Fe(II) by the P-450 system; (b) Fe(II) and H2O2 (anaerobically), in the absence of a P-450 system, catalyze glutamine synthetase inactivation; (c) inactivation is inhibited by catalase; and (d) hexobarbital, which stimulates the rate of H2O2 production by the P-450 system, stimulates the rate of glutamine synthetase inactivation. Moreover, inactivation of glutamine synthetase by the P-450 system does not require complex formation because inactivation occurs when the P-450 components and the glutamine synthetase are separated by a semipermeable membrane. Also, if endogenous catalase is inhibited by azide, rabbit liver microsomes catalyze the inactivation of glutamine synthetase.     

The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase.

The essential gene efg, which complements ammonia-dependent growth (adgA) mutations in Rhodobacter capsulatus and is located at 38.1 min on the Escherichia coli chromosome, was found to code for NH3-dependent NAD synthetase. Crude extracts from a strain which overproduces the efg gene product contained up to 400 times more activity than crude extracts from the control strain, and the purified Efg protein possessed-NH3-dependent NAD synthetase activity. Glutamine-dependent NAD synthetase activity was found in crude extracts of E. coli but not in the purified enzyme, suggesting that it may be catalyzed by an additional subunit. An R. capsulatus strain carrying an adgA mutation was found to be deficient in NAD synthetase activity, and activity was restored by complementation with the E. coli gene. In accordance with the nomenclature proposed for Salmonella typhimurium (K. T. Hughes, B. M. Olivera, and J. R. Roth, J. Bacteriol. 170:2113-2120, 1988), the efg and adgA genes should now be designated nadE.     

Complementation of DNA repair-deficient Escherichia coli by the yeast Apn1 apurinic/apyrimidinic endonuclease gene

The Saccharomyces cerevisiae APN1 gene encoding an AP endonuclease/3'-diesterase was engineered in vitro for expression in Escherichia coli. The expression vector directs the synthesis in E. coli of a Mr 40,500 protein that reacts with anti-Apn1 antibodies and has the DNA-repair activities characteristic of Apn1 isolated from yeast. A band corresponding to Apn1 was observed in DNA repair activity gels only with extracts of E. coli harbouring the APN1 expression plasmid. Expression of Apn1 conferred resistance to oxidants and alkylating agents in E. coli lacking exonuclease III and endonuclease IV. For H2O2 damage, this rescue effect was correlated with the repair of oxidative lesions in the bacterial chromosome by the Apn1 protein. Thus, Apn1 can function in bacteria in a manner similar to its proposed multiple functions in yeast.     

Rapid method for altering bacterial ribosome-binding sequences for overexpression of proteins in Escherichia coli.

In an Escherichia coli expression system, two genes, one from an anaerobic intestinal bacterium and one from E. coli, were overexpressed following the alteration of ribosome-binding (Shine-Dalgarno) sequences. For both genes, the polymerase chain reaction (PCR) was used to modify the ribosome-binding sequence and, at the same time, provide restriction endonuclease sequences at each end of the gene. These restriction endonuclease sequences were used for inserting the DNA into the E. coli plasmid vector pGEM2, which has the T7 promoter upstream from its multiple cloning sites. Each chimeric plasmid, made by ligating the PCR product into pGEM2, was transformed into E. coli strain HMS174(DE3) which, when induced, produces T7 RNA polymerase for regulated overexpression. The gene isolated from the anaerobic intestinal bacterium, a 27-kDa polypeptide gene from Eubacterium sp. strain 12708, when expressed using this system, produced about one-third of the total cell protein as measured in Coomassie-stained protein gels and confirmed by Western blots with rabbit antibody. The E. coli enzyme, a 28.4-kDa tRNA methylation enzyme, was increased fivefold in activity of cell extracts over that of the best previous strain.     

An activity stain for dihydroxy-acid dehydratase

An activity stain has been devised for the dihydroxy-acid dehydratase. When applied to polyacrylamide gel electropherograms of crude soluble extracts of Escherichia coli, it detected a single electromorph. The intensity of staining increased with the amount of extract protein applied to the gel. Activity staining demonstrated that (a) anaerobically grown cells contain more extractable dehydratase activity than do aerobically grown cells; (b) exposure of E. coli to 4.2 atm O2 caused virtually complete loss of activity; (c) exposure of cells to paraquat or plumbagin in the presence of dioxygen, but not in its absence, caused a massive loss of activity. These data illustrate the utility of this activity stain and demonstrate that the dehydratase is inactivated by O2- generated within cells.     

Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA

The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.     

Superoxide dismutase and O2 lethality in Bacteroides fragilis.

Exposure of midlog Bacteroides fragils (VPI 2393) to 2% O2-98% N2 caused a three- to fivefold increase in superoxide dismutase specific activity within the cells. The increase in specific activity was completed within 90 min after exposure to oxygen and was dependent upon protein synthesis. Cells containing the higher superoxide dismutase level were more resistant to the effects of 5 atm of oxygen tension than were cells containing the lower level of superoxide dismutase but were equally resistant to 5 atm of nitrogen tension. Similar results were observed upon comparing viability experiments with B. fragilis and B. vulgatus. Superoxide dismutase activity in sonic extracts of B. fragilis was rapidly inactivated by exposure to 5 mM H2O2 and was inhibited by 1 mM NaN3 but not 5 mM NaCN. The inhibition pattern is identical to the pattern demonstrated for the purified iron-containing enzyme from Escherichia coli B and suggests that the superoxide dismutase in B. fragilis is an iron enzyme.     

Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.     

CMP-N-acetylneuraminic acid synthetase of Escherichia coli: high level expression, purification and use in the enzymatic synthesis of CMP-N-acetylneuraminic acid and CMP-neuraminic acid derivatives.

The gene encoding CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) in Escherichia coli serotype O7 K1 was isolated and overexpressed in E.coli W3110. Maximum expression of 8-10% of the soluble E.coli protein was achieved by placing the gene with an engineered 5'-terminus and Shine-Dalgarno sequence into a pKK223 vector derivative behind the tac promoter. The overexpressed synthetase was purified to greater than 95% homogeneity in a single step by chromatography on high titre Orange A Matrex dye resin. Enzyme purified by this method was used directly for the synthesis of CMP-NeuAc and derivatives. The enzymatic synthesis of CMP-NeuAc was carried out on a multigram scale using equimolar CTP and N-acetylneuraminic acid as substrates. The resultant CMP-NeuAc, isolated as its disodium salt by ethanol precipitation, was prepared in an overall yield of 94% and was judged to be greater than 95% pure by 1H NMR analysis. N-Carbomethoxyneuraminic acid and N-carbobenzyloxyneuraminic acid were also found to be substrates of the enzyme; 5-azidoneuraminic acid was not a substrate of the enzyme. N-Carbomethoxyneuraminic acid was coupled to CMP at a rate similar to that observed with NeuAc, whereas N-carbobenzyloxyneuraminic acid was coupled greater than 100-fold more slowly. The high level of expression achieved with the E.coli synthetase, together with the high degree of purity readily obtainable from crude cell extracts, make the recombinant bacterial enzyme the preferred catalyst for the enzymatic synthesis of CMP-N-acetylneuraminic acid.