ABSTRACT
Polysialic acids (PSA) are important extracellular virulence factors of the human pathogens Neisseria meningitidis and Escherichia coli. The importance of these polysaccharides in virulence make the polysialyltransferases (PST) targets for therapeutic drugs and protein engineering to facilitate efficient vaccine production. Here, we have generated recombinant bovine nucleotide monophosphate kinase to facilitate steady state kinetic assays of the PST. We have characterized the N. meningitidis group C (NmC) PST kinetically, using substrate analogues to describe the polymerization reaction. We observed a decrease in Km as the length of the oligo-sialic acid acceptor was increased, indicating a tighter binding of longer oligomers. In addition, we observed a biphasic relationship between kcat and chain length, which can be attributed to a switch in the mechanism of transfer of sialic acid from distributive to processive as the chain length increased above six sialic acid units. Substitution of donor substrate with the analogue CMP-9-F-sialic acid had minimal effect on acceptor Km, but it decreased kcat 6-fold. We propose that this decrease in kcat is caused by a destabilization of the transition state and/or an increase affinity of the product due to presence of the fluoro substituent. The acceptor's hydrophobicity also plays a role in catalysis. The kinetic analysis of the NmC PST with hydrophobic aglycon acceptor substrates indicated that they bind tighter and are turned over at a faster rate than the α-2,9 polysialic acid substrates lacking the hydrophobic end. This finding suggests the presence of a secondary ligand binding site that tethers the acceptor substrate to the enzyme active site.
