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.     

Novel method for enzymatic synthesis of CMP-NeuAc

A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.     

Molecular modeling and dynamics studies of cytidylate kinase from Mycobacterium tuberculosis H37Rv

Bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) catalyses the phosphoryl transfer from ATP to CMP and dCMP, resulting in the formation nucleoside diphosphates. In eukaryotes, CMP/UMP kinase catalyses the conversion of UMP and CMP to, respectively, UDP and CDP with high efficiency. This work describes for the first time a model of bacterial cytidylate kinase or cytidine monophosphate kinase (CMP kinase) from mycobacterium tuberculosis (MtCMPK). We modeled MtPCMPK in apo form and in complex with cytidine 5′-monophosphate (CMP) to try to determine the structural basis for specificity. Comparative analysis of the model of MtCMPK allowed identification of structural features responsible for ligand affinities. Analysis of the molecular dynamics simulations of these two systems indicates the structural features responsible for the stability of the structure, and may help in the identification of new inhibitors for this enzyme.     

Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose

Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically fixed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mM UMP.     

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.     

Substrate-induced fit of the ATP binding site of cytidine monophosphate kinase from Escherichia coli: time-resolved fluorescence of 3'-anthraniloyl-2'-deoxy-ADP and molecular modeling

The conformation and dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichia coli (CMPK(coli)), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the enzyme. The spectroscopic properties of the bound fluorescent nucleotide change strongly with respect to those in aqueous solution. These changes (red shift of the absorption and excitation spectra, large increase of the excited state lifetime) are compared to those observed in different solvents. These data, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected from the aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP. The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completely blocked by CMP binding. The energy-minimized models of CMPK(coli) complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes and, respectively) were compared to the crystal structure of UMP/CMP kinase from Dictyostelium discoideum complexed with substrates (PDB code ). The key residues for ATP/ADP binding to CMPK(coli) were identified as R157 and I209, their side chains sandwiching the adenine ring. Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins. In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LID conformation remained unchanged, by contrast to the fluorescence data. These results provide the first experimental arguments about an ATP-mediated induced-fit of the LID in CMPK(coli) modulated by CMP, leading to a closed conformation of the active site, protected from water.     

One-pot enzymatic synthesis of UDP-D-glucose from UMP and glucose-1-phosphate using an ATP regeneration system

Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.     

Solution structure and function of an essential CMP kinase of Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity and has developed resistance to many antibiotics. We show that the gene product from SP1603, identified from S. pneumoniae TIGR4, is a CMP kinase that is essential for bacterial growth. It represents an attractive drug target for the development of a novel antibiotic to overcome the problems of drug resistance development for this organism. Here we describe the three-dimensional solution structure of the S. pneumoniae CMP kinase as determined by NMR spectroscopy. The structure consists of eight alpha-helices and two beta-sheets that fold into the classical core domain, the substrate-binding domain, and the LID domain. The three domains of the protein pack together to form a central cavity for substrate-binding and enzymatic catalysis. The S. pneumoniae CMP kinase resembles the fold of the Escherichia coli homolog. An insertion of one residue is observed at the beta-turn in the substrate-binding domain of the S. pneumoniae CMP kinase when compared with the E. coli homolog. Chemical shift perturbations caused by the binding of CMP, CDP, and ATP revealed that CMP or CDP binds to the junction between the core and substrate-binding domains, whereas ATP binds to the junction between the core and LID domains. From NMR relaxation studies, we determined that the loops in the LID domain are highly mobile. These mobile loops could aid in the closing/opening of the LID domain during enzyme catalysis.     

Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.     

Prolonging cell-free protein synthesis with a novel ATP regeneration system

A new approach for the regeneration of adenosine triphosphate (ATP) during cell-free protein synthesis was developed to prolong the synthesis and also to avoid the accumulation of inorganic phosphate. This approach was demonstrated in a batch system derived from Escherichia coli. Contrary to the conventional methods in which exogenous energy sources contain high-energy phosphate bonds, the new system was designed to generate continuously the required high-energy phosphate bonds within the reaction mixture, thereby recycling the phosphate released during protein synthesis. If allowed to accumulate, phosphate inhibits protein synthesis, most likely by reducing the concentration of free magnesium ion. Pediococcus sp. pyruvate oxidase, when introduced in the reaction mixture along with thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD), catalyzed the generation of acetyl phosphate from pyruvate and inorganic phosphate. Acetyl kinase, already present with sufficient activity in Escherichia coli S30 extract, then catalyzed the regeneration of ATP. Oxygen is required for the generation of acetyl phosphate and the H(2)O(2) produced as a byproduct is sufficiently degraded by endogenous catalase activity. Through the continuous supply of chemical energy, and also through the prevention of inorganic phosphate accumulation, the duration of protein synthesis is extended up to 2 h. Protein accumulation levels also increase. The synthesis of human lymphotoxin receives greater benefit than than that of chloramphenicol acetyl transferase, because the former is more sensitive to phosphate inhibition. Finally, through repeated addition of pyruvate and amino acids during the reaction period, protein synthesis continued for 6 h in the new system, resulting in a final yield of 0.7 mg/mL.     

Phosphorylation of recombinant human ATP:citrate lyase by cAMP-dependent protein kinase abolishes homotropic allosteric regulation of the enzyme by citrate and increases the enzyme activity. Allosteric activation of ATP:citrate lyase by phosphorylated sugars

Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.     

Enzymatic production of (R)-phenylacetylcarbinol by pyruvate decarboxylase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Herein, we synthesized (R)-phenylacetylcarbinol (PAC), a pharmaceutical intermediate for ephedrine and pseudoephedrine, from benzaldehyde and pyruvate using a recombinant pyruvate decarboxylase (PDC) from Zymomonas mobilis. A whole cell reaction consisting of 30 mM benzaldehyde, 60 mM pyruvate, and a mutant PDC enzyme (PDC W329M; 1.6 mg DCW/mL) produced 12.4 mM (R)-PAC and less than 0.3 mM benzyl alchohol in 15 h at 20°C, outperforming the crude enzyme extract reaction (1.2 mM (R)-PAC) and minimizing formation of benzyl alchohol, the major by-product of S. cerevisiae whole cell reaction. These observations suggested that recombinant E. coli whole cell reactions are more efficient than crude enzyme extract or yeast-based reactions. We also demonstrated that the E. coli whole cell reaction performed effectively without expensive thiamin diphosphate cofactor. Finally, whole cell reaction (8 mg DCW/mL) was carried out with 200 mM benzaldehyde, 400 mM pyruvate in 10 mL of 500 mM phosphate buffer (pH 6.5), and 72 mM (R)-PAC was produced with 36% conversion for 15 h. © KSBB     

2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase

A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells

The Saccharomyces cerevisiae cell wall provides a semipermeable barrier that can retain intracellular proteins but still permits small molecules to pass through. When S. cerevisiae cells expressing E. coli lacZ are treated with detergent to extract the cell membrane, beta-galactosidase activity in the permeabilized cells is approximately 40% of the activity of the protein in cell extract. However, the permeabilized cells can easily be collected and reused over 15 times without appreciable loss in activity. Cell wall composition and thickness can be modified using different cell strains for enzyme expression or by mutating genes involved in cell wall biosynthesis or degradation. The Sigma1278b strain cell wall is less permeable than the walls of BY4742 and W303 cells, and deleting EXG1, which encodes a 1,3-beta-glucanase, can further reduce permeability. A short Zymolyase treatment can increase cell wall permeability without rupturing the cells. Encapsulating multiple enzymes in permeabilized cells can offer kinetic advantages over the same enzymes in solution. Regeneration of ATP from AMP by adenylate kinase and pyruvate kinase encapsulated in the same cell proceeded more rapidly than regeneration using a cell extract. Combining permeabilized cells containing adenylate kinase with permeabilized cells containing pyruvate kinase can also regenerate ATP from AMP, but the kinetics of this reaction are slower than regeneration using cell extract or permeabilized cells expressing both enzymes.     

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5′-nucleotidase deficiency

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP:d-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5′-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di-, and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl₂ to promote PRPP synthetase subunit aggregation. Increasing the MgCl₂ to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.     

Catalytic properties of Escherichia coli polyphosphate kinase: an enzyme for ATP regeneration.

Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.     

Purification and properties of N-acetylneuraminate lyase from Escherichia coli

N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4.1.3.3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase. The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60 degrees C. The thermal stability increased up to 75 degrees C in the presence of pyruvate. No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as N-bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromercuribenzoic acid and mercuric chloride were also potent inhibitors. The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6 mM and 4.3 mM, respectively. Pyruvate inhibited the cleavage reaction competitively; Ki was calculated to be 1.0 mM. In the condensation reaction, N-acetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, beta-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate. The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits. The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.     

Enterococcus faecalis phosphomevalonate kinase

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.