Bacterial sialidases represent important colonization
or virulence factors. The development of a rational
basis for the design of antimicrobials targeted to sialidases
requires the knowledge of the exact roles of
their conserved amino acids. A recombinant enzyme
of the small (43 kDa) sialidase of Clostridium perfringens
was used as a model in our study. Several conserved
amino acids, identified by alignment of known
sialidase sequences, were altered by sitedirected
mutagenesis. All recombinant enzymes were affinitypurified
and the enzymatic characteristics were determined.
Among the mutated enzymes with modifications
in the environment of the 4-hydroxyl group of
bound sialic acids, D54N and D54E exhibited minor
changes in substrate binding. However, a reduced activity
and changes in their pH curves indicate the importance
of a charged group at this area. R56K, which
is supposed to bind directly to sialic acids as in the
homologous Salmonella typhimurium sialidase,
showed a 2500-fold reduced activity. The amino acids
Asp-62 and Asp-100 are probably involved in catalysis,
indicated by reduced activities and altered temperature
and pH curves of mutant enzymes. Exchanging
Glu-230 with threonine or aspartic acid led
to dramatic decreases in activity. This residue and Y
347 are supposed to be crucial for providing a suitable
environment for catalysis. However, unaltered
pH curves of mutant sialidases exclude their direct involvement
in protonation or deprotonation events.
These results indicate that the interactions with the
substrates vary in different sialidases and that they might be more complex than suggested by mere
static Xray structures.
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