Cloning, over-expression, purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae.
N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues.
Suryanti
Venty
V
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
Nelson
Adam
A
Berry
Alan
A
D000645:Ammonium Sulfate / Q000494:pharmacology; D001412:Bacillus subtilis / Q000201:enzymology; D002384:Catalysis; D002845:Chromatography; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019992:Databases as Topic; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006595:Hexosamines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008670:Metals / Q000494:pharmacology; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010658:Phenylglyoxal / Q000494:pharmacology; D010728:Phosphoenolpyruvate / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D013292:Streptococcus agalactiae / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D017392:Thiobarbituric Acid Reactive Substances; D013997:Time Factors
D006595:Hexosamines; D007477:Ions; D008670:Metals; D017392:Thiobarbituric Acid Reactive Substances; D010728:Phosphoenolpyruvate; D004492:Edetic Acid; C018730:N-acetylneuraminate synthase; D007652:Oxo-Acid-Lyases; D019158:N-Acetylneuraminic Acid; D010658:Phenylglyoxal; D000645:Ammonium Sulfate; C002022:N-acetylmannosamine