title: Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens: insights into the reaction mechanism.
abstract: The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography. The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 degrees C. In kinetic studies the lyase exhibits a Km of 3.2 mM for Neu5Ac and a Vmax of 27.5 U x mg(-1). To clarify the functional role of some putative active site residues, site-directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate. Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding. In this respect the carboxy group of aspartate 187 seems to be particularly important. Based on the results of these studies, a model of the reaction mechanism is discussed.
authors: Biochemisches Institut, Christian-Albrechts-Universität, Kiel, Germany.
authors: D
authors: D
authors: Krüger
authors: R
authors: R
authors: Schauer
authors: C
authors: C
authors: Traving
mesh_terms: D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase