Effects of site-specific mutations on the enzymatic properties of a sialidase from Clostridium perfringens.

Three site-specific mutations were performed in two regions of a sialidase gene from Clostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed in Escherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, as KM and Vmax, as well as K(i) measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.