2Institute of Molecular Biology and Biochemistry, Free University of Berlin, Arnimallee 22, 14195 Berlin-Dahlem, Germany, and 3College of Life Science, Nanjing University, Nanjing 210093, Peoples Republic of China
Received on June 1, 2001; revised on September 6, 2001; accepted on September 17, 2001.
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Abstract |
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Key words: N-acetylmannosamine-6-phosphate/ N-acetylneuraminic acid-9-phosphate synthase/phosphoenolpyruvate/sialic acid
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Introduction |
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N-Acetylneuraminic acid (Neu5Ac), the predominant sialic acid in nature, is synthesized in vivo by a multistep cytosolic pathway. In mammals this starts with the formation of N-acetylmannosamine (ManNAc) from UDP-N-acetylglucosamine (UDP-GlcNAc) by the action of UDP-GlcNAc 2-epimerase. ManNAc is then phosphorylated at C-6 by a specific kinase. Recently it was found that these two steps are catalyzed by one bifunctional enzyme (Hinderlich et al., 1997; Stäsche et al., 1997
). ManNAc-6-phosphate is then converted to Neu5Ac-9-phosphate by condensation with phosphoenolpyruvate (PEP) catalyzed by Neu5Ac-9-phosphate synthase (Watson et al., 1966
). The phosphate is released, probably by a specific phosphatase (Jourdian et al., 1964
), and finally CMP-Neu5Ac, the activated Neu5Ac donor for glycolipid and glycoprotein oligosaccharide biosynthesis, is formed in the nucleus (Kean, 1970
).
Neu5Ac-9-phosphate synthase was first described in hog submaxillary glands (Roseman et al., 1961) and rat liver (Warren and Felsenfeld, 1961
). The specifity of the enzyme for ManNAc-6-phosphate and PEP was shown for the purified hog enzyme (Watson et al., 1966
). Watson et al. (1966)
and Van Rinsum et al. (1984)
detected the enzyme in all rat tissues investigated with the highest activity in salivary glands. In contrast to the mammalian enzyme, the bacterial enzyme catalyzes the condensation of ManNAc and PEP in the synthesis of Neu5Ac (Vann et al., 1997
). It was also suggested that the bacterial Neu5Ac lyase synthesizes sialic acid by condensation of ManNAc and pyruvate (Ferrero et al., 1995
; Rodriguez-Aparicio et al., 1995
). Nevertheless it was possible to clone the human (Lawrence et al., 2000
) and murine (Nakata et al., 2000
) cDNA of Neu5Ac-9-phosphate synthase based on homology to the Escherichia coli Neu5Ac synthase gene (neuB). In this article we report the purification of Neu5Ac-9-phosphate synthase to homogeneity and the characterization of its biochemical properties.
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Results |
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Sequence analysis
To verify that the 37-kDa polypeptide found in SDSPAGE is Neu5Ac-9-phosphate synthase, a part of the amino acid sequence was determined. N-Terminal sequencing was not successful, indicating a blocked N-terminus. Therefore the protein was digested by trypsin and the masses of the resulting peptides were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Four of the peptides had the same masses as theoretical tryptic peptides derived from amino acid sequences of murine Neu5Ac-9-phosphate synthase (Table II). For control purposes peptide 1 was analyzed by the postsource decay method, and its sequence was identical to the corresponding peptide of the mouse enzyme. Therefore we conclude that the 37-kDa polypeptide represented rat Neu5Ac-9-phosphate synthase.
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The product of the reaction of Neu5Ac-9-phosphate synthase was characterized with the aid of radiolabeled compounds. Purified enzyme was assayed under standard conditions with addition of [14C]-ManNAc 6-phosphate. The compounds were separated by paper chromatography. The product of the enzymatic reaction was Neu5Ac-9-phosphate and no formation of Neu5Ac was observed (data not shown). Therefore it can be concluded that Neu5Ac-9-phosphate synthase does not have an additional Neu5Ac-9-phosphatase activity.
Next we substituted ManNAc-6-phosphate with other sugar phosphates (GlcN-6-phosphate, Frc-6-phosphate, GlcNAc-1-P, GlcNAc-6-phosphate) and with ManNAc. Additionally, PEP was replaced by pyruvate. No enzyme activity was observed with any of these substrates, indicating a high specificity of Neu5Ac-9-phosphate synthase for ManNAc-6-phosphate and PEP. The enzyme showed Km values of 35 µM for ManNAc-6-phosphate and 105 µM for PEP. In contrast to the findings of Watson et al. (1966) for hog submaxillary gland Neu5Ac-9-phosphate synthase, which displayed a nonlinear progression for PEP in a [S]/v versus [S] plot, the Lineweaver-Burk plot for the rat enzyme showed a linear progression. This may indicate that exclusively the hog enzyme is regulated by cooperativity.
Neu5Ac-9-phosphate synthase activity depended on the concentration of Mg2+, the physiological divalent cation in the cytosol. Maximum activity was observed as a Mg2+ concentration of 5 mM (data not shown), in agreement with the findings of Watson et al. (1966) for the hog submaxillary gland enzyme. Mg2+ ions could be replaced by other divalent cations, such as Mn2+, Fe2+, or Ni2+. At 5 mM, Mn2+, Fe2+, and Ni2+ showed 188%, 122%, and 62%, respectively, of the activity of Mg2+. Enzyme activity in the presence of 5 mM Cu2+ and Ca2+, respectively, was less than 5%, therefore these cations were not able to replace Mg2+.
Inactivation of Neu5Ac-9-phosphate synthase by cysteine-specific chemical modifiers
Putative essential amino acid residues in the active site of Neu5Ac-9-phosphate synthase were identified with the aid of specific chemical modifiers. Modification of lysines by 2,4,6-trinitrobenzenesulfonic acid and arginines by phenylglyoxal and histidines by diethylpyrocarbonate had no influence on enzyme activity (data not shown). On the other hand, the cysteine modifiers N-ethylmaleimide and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) were very effective inhibitors of Neu5Ac-9-phosphate synthase activity. N-Ethylmaleimide (1 µM) inactivated the enzyme completely after 30 min. DTNB was even more effective. A 10-min incubation with 0.3 µM DTNB had no effect, presumably due to the reaction of DTNB with traces of redox reagents in the buffer, but in the range of 0.3 µM to 0.8 µM a clear concentration dependency was observed (data not shown). DTNB at 0.8 µM was sufficient for complete enzyme inhibition. These data show that at least one highly reactive cysteine is located in or near the active site of Neu5Ac-9-phosphate synthase.
Interestingly, the inhibitory effect of DTNB was positively influenced by Mg2+ in a concentration-dependent manner. An incubation of Neu5Ac-9-phosphate synthase with 0.6 µM DTNB for 30 min in the absence of Mg2+ resulted in a residual activity of 35%. The identical assay in the presence of 5 mM Mg2+ showed a residual enzyme activity of only 20%. The effect was amplified by increasing concentrations of Mg2+ (data not shown). Assuming that Mg2+ is not required for the modification reaction, this indicates that Mg2+ has an allosteric effect on the structure of the active site of the enzyme and exposes the cysteine(s) more effectively to DTNB.
To localize the active site cysteine(s) in more detail, the enzyme was preincubated with its substrates before DTNB treatment. Whereas ManNAc-6-phosphate did not protect the enzyme against inhibition, PEP partially protected the cysteine(s) against modification (Figure 3). Addition of Mg2+ to the assay increased the protection effect of PEP, but no further effect was observed by addition of ManNAc 6-phosphate. Therefore we conclude that the active site cysteine(s) play an important role in binding PEP or a PEP/Mg2+ complex.
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Discussion |
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To confirm that the 37-kDa polypeptide represents rat Neu5Ac-9-phosphate synthase, it was tryptically digested and the resulting peptides were analyzed by MALDI-TOF MS. Four peptides corresponded exactly to theoretical tryptic peptides of the murine enzyme, indicating a high sequence similarity of the two proteins. Additionally, the human and mouse enzymes show 93% sequence similarity (Lawrence et al., 2000; Nakata et al., 2000
), suggesting that mammalian enzymes are generally closely related. On the other hand, human Neu5Ac-9-phosphate synthase differs from the murine enzyme in that it can use Man-6-phosphate as a substrate to synthesize KDN-9-phosphate (Lawrence et al., 2000
). KDN is a sialic acid sharing many features with Neu5Ac, occurring in glycoconjugates and displaying variations in the
-ketosidic linkage to the penultimate sugar residue (Inoue et al., 1996
). We found no KDN-9-phosphate synthase activity for the rat Neu5Ac-9-phosphate synthase. Admittedly, we used a thiobarbituric acid assay for detection of sialic acids, which was about 10-fold less sensitive than the high-performance liquid chromatography (HPLC) analysis used by Lawrence et al. (2000)
, but Nakata et al. (2000)
used HPLC also for the analysis of KDN-9-phosphate synthase activity of the murine enzyme and ruled it out completely. This indicates that a separate KDN-9-phosphate synthase, which has already been found in trout testis (Angata et al., 1999
), has to be postulated at least for rodents. Site-directed mutagenesis of amino acids not conserved between rodent and human Neu5Ac-9-phosphate synthase may be used to engineer the rodent enzymes to a human enzyme or vice versa.
Synthesis of Neu5Ac is strongly regulated by the initial enzyme of the pathway, UDP-GlcNAc 2-epimerase/ManNAc kinase (Hinderlich et al., 1997), by a CMP-Neu5Ac-mediated feed back inhibition of the epimerase activity (Kornfeld et al., 1964
). We analyzed the Neu5Ac-9-phosphate synthase for regulatory mechanisms. Dependency of the enzymatic reaction on substrate concentrations fitted perfectly with Michaelis-Menten kinetics, indicating no regulation by cooperativity effects. Potential allosteric effectors like CMP-Neu5Ac or UDP-GlcNAc had also no effect on enzymatic activity (data not shown). Nevertheless, Neu5Ac-9-phosphate synthase activity may be coupled directly to the regulation of UDP-GlcNAc 2-epimerase/ManNAc kinase, by the availability of the substrate ManNAc-6-phosphate. Expression of two separate synthases for KDN-9-phosphate and Neu5Ac-9-phosphate favors a more coordinated and separately regulated biosynthesis of the two sialic acids.
Divalent cations are essential for the activity of Neu5Ac-9-phosphate synthase. Stephens and Bauerle (1992) showed, for the related E. coli 3-deoxy-arabino-heptulosonate 7-phosphate synthase, that an active site cysteine is directly involved in binding of the cation and the substrate PEP. We showed that the cysteine-specific chemical modifier, DTNB, completely inhibited Neu5Ac-9-phosphate synthase activity, and that optimal protection against inactivation was provided by a PEP/Mg2+ complex. Furthermore, a cysteine serves as a nucleophile in C-O bond cleavage at PEP during the catalytic process of E. coli 3-deoxy-manno-octulonate 8-phosphate synthase (Hedstrom and Abeles, 1988
). A general role of cysteines in the mechanism of the aldol addition of the sugar and PEP is therefore suggested. Both mouse and human Neu5Ac-9-phosphate synthase contain eight cysteine residues. Due to the very high sequence homology of the mammalian enzymes, this should also count for rat Neu5Ac-9-phosphate synthase. An aligment with several related bacterial Neu5Ac synthases (Figure 4) showed only one highly conserved cysteine residue among the species. This strongly favours mammalian Cysteine-184 as the catalytically important one. A site-directed mutagenesis study may clarify this in the future.
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Materials and methods |
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Neu5Ac-9-phosphate synthase assay
Neu5Ac-9-phosphate synthase activity was assayed by a modification of the method of Warren (1959). In brief, incubation mixtures contained 50 mM Na2HPO4, pH 7.5, 12.5 mM MgCl2, 8.3 mM PEP, 3.3 mM ManNAc-6-phosphate, and various amounts of enzyme solution in a final volume of 125 µl. Incubations were carried out at 37°C for 30 min and stopped by heating at 100°C for 3 min. After centrifugation for 3 min at 13,000 x g, 137 µl of periodic acid solution (2.5 mg/ml in 57 mM H2SO4) were added and incubated for 15 min at 37°C. Then 50 µl sodium arsenite solution (25 mg/ml in 0.5 M HCl) were added, and the tubes were shaken vigorously to ensure complete elimination of the yellow-brown color. After this step 100 µl of 2-thiobarbituric acid solution (71 mg/ml adjusted to pH 9.0 with NaOH) were added, and the samples were heated to 100°C for 7.5 min. The solution was extracted with 1 ml of butanol/5% 12 M HCl, and the phases were separated by centrifugation. The absorbance of the organic phase was measured at 549 nm.
The radiolabeled assay was performed as described in the previous paragraph with addition of 50 nCi [14C]-ManNAc-6-phosphate. The assay was stopped by addition of 200 µl ethanol. Radiolabeled compounds were separated by descending paper chromatography as described by Zeitler et al. (1992). The respective Rf values were 0.08 for Neu5Ac-9-phosphate, 0.17 for ManNAc-6-phosphate, and 0.24 for Neu5Ac.
Protein concentration was measured by the method of Bradford (1976), using bovine serum albumin as standard. One unit of enzyme activity was defined as the formation of 1 µmol of Neu5Ac-9-phosphate per min at 37°C. Specific activity was expressed as mU per mg of protein.
Purification of Neu5Ac-9-phosphate synthase
Step 1: Preparation of rat liver cytosol.
Male Wistar rat livers were removed under light ether anesthesia and transferred to 2 volumes of homogenizing buffer containing 10 mM NaH2PO4, pH 7.5, 1 mM ethylenediamine tetra-acetic acid, 1 mM dithiothreitol (buffer A), and 1 mM phenylmethylsulfonyl fluoride. The tissue was homogenized with an Ultraturrax for 1.5 min at 10,000 rpm. The homogenate was ultracentrifuged at 100,000 x g for 60 min.
Step 2: Ammonium sulfate precipitation.
The supernatant from step 1 was fractionated with 1.5 M and 3.5 M ammonium sulfate, respectively. The precipitates were collected by centrifugation at 20,000 x g for 20 min. The pellet from the second centrifugation was dissolved in buffer A, pH 7.0, and centrifuged at 30,000 x g for 10 min.
Step 3: Desalting on Sephadex G-25.
The supernatant from step 2 was applied to a column (26 mm ID x 60 cm) of Sephadex G-25. The column was eluted with buffer A, pH 7.0, and the protein fraction with absorbance at 280 nm was collected.
Step 4: Hydroxylapatite chromatography.
The fraction from step 3 was applied to a column (26 mm ID x 20 cm) of hydroxylapatite, equilibrated with buffer A, pH 7.0. The column was eluted with 130 ml of a linear gradient starting with buffer A, pH 7.0, and finishing with 200 mM NaH2PO4, pH 7.0, 1 mM ethylenediamine tetra-acetic acid, 1 mM dithiothreitol. Fractions were collected at a flow rate of 1 ml/min.
Step 5: Hydrophobic interaction chromatography on phenyl-Sepharose.
Fractions containing enzyme activity from step 4 were adjusted to 3 M NaCl and then applied to a column (26 mm ID x 20 cm) of phenyl-Sepharose. The column was washed with 100 ml of buffer A containing 3.0 M NaCl. The enzyme was eluted with 140 ml of a linear gradient of 3 M to 0 M NaCl in buffer A at a flow rate of 1 ml/min.
Step 6: Anion exchange chromatography on MonoQ.
Fractions with enzyme activity from step 5 were desalted (see step 3) and then applied to a MonoQ HR 10/10 column. The column was washed with 25 ml buffer A and eluted with 50 ml of a linear gradient of 0 M to 0.25 M NaCl. Fractions were collected at a flow rate of 0.5 ml/min.
Step 7: Gel filtration chromatography on Superdex 200.
Fractions with the highest enzyme activities from step 6 were concentrated to 0.3 ml on UH 100/25 Ultra Thimbles (Schleicher & Schuell, Germany), then applied to a Superdex 200 HR 10/30 column. The column was equilibrated with buffer A and eluted with the same buffer. The fractions were collected at a flow rate of 0.25 ml/min.
All procedures were carried out at 04°C. Fractions obtained during purification were assayed for Neu5Ac-9-phosphate synthase activity as described in Neu5Ac-9-phosphate synthase assay, and analyzed for purity by SDSPAGE according to the method of Laemmli (1970). Gels were silver stained for protein as described elsewhere (Heukeshoven and Dernick, 1985
).
Peptide MS
Prior to digestion, 10 µg of purified Neu5Ac-9-phosphate synthase were treated with 55 mM iodacetamide for 20 min in the dark at room temperature. The protein was then digested with 0.2 µg of trypsin in 50 mM ammonium bicarbonate and 5 mM CaCl2 at 37°C for 16 h. The MS analysis of proteolytically derived peptides was performed using a Bruker Reflex MALDI-TOF mass spectrometer with -cyano-4-hydroxycinnimanic acid as a matrix. For the analysis of fragment ions generated by postsource decay (Kaufmann et al., 1993
) the FASTTM method developed by Bruker was used.
Kinetic analysis
Kinetic data were determined for Neu5Ac-9-phosphate synthase purified as described in Purification of Neu5Ac-9-phosphate synthase. The enzyme activity assay contained 50 mM Na2HPO4, pH 7.5, 12.5 mM MgCl2, a variable concentration of PEP in the presence of 3.3 mM ManNAc-6-phosphate, or a variable concentration of ManNAc-6-phosphate in the presence of 8.3 mM PEP. Michaelis constants (Km) were determined by Lineweaver-Burk plots.
Effect of divalent cations
Neu5Ac-9-phosphate synthase from the final purification step was assayed in 50 mM NaH2PO4, pH 7.5, 1 mM dithiothreitol, 8.3 mM PEP, 3.3 mM ManNAc-6-phosphate after addition of the following salts (7 mM, if not otherwise indicated): MgCl2, CaCl2, MnCl2, FeSO4, NiCl2, and CuCl2.
Inactivation of Neu5Ac-9-phosphate synthase by chemical modifiers
Dithiothreitol-free enzyme was freshly prepared from MonoQ fractions by gel filtration as described under purification step 7, with the exception that dithiothreitol was removed from the elution buffer. Modification was performed by incubating Neu5Ac-9-phosphate synthase with the indicated amounts of different modifiers. Extent of inactivation was monitored by measuring residual enzyme activities in aliquots withdrawn from the incubation mixture and from control experiments without modifier at the indicated times. All inactivation experiments were carried out at 37°C.
The enzyme was protected against inactivation by incubation with 8.3 mM of PEP, 3.3 mM ManNAc-6-phosphate, or 5 mM MgCl2 before adding the modifier. Inactivation and corresponding control experiments were carried out and monitored as described.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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