Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens - Insights into the reaction mechanism

The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homoge neity using metal chelate affinity and anion exchange chromatography. The p urified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 d egreesC. In kinetic studies the lyase exhibits a K-m of 3.2 mM for Neu5Ac a nd a V-max of 27.5 U.mg(-1). To clarify the functional role of some putativ e active site residues, site-directed mutagenesis was performed. Lysine 161 was identified as the residue forming the Schiff base intermediate with th e substrate. Tyrosine 133 was shown to be also a catalytically important re sidue; it seems to function as an acceptor for the proton of the C-4 hydrox yl group, as already suggested by other groups. Furthermore, it is involved in stabilizing the Schiff base intermediate. Mutations of aspartate 187 an d glutamate 188 indicate that both residues are involved in substrate bindi ng. In this respect the carboxy group of aspartate 187 seems to be particul arly important. Based on the results of these studies, a model of the react ion mechanism is discussed.