Kinetic characterization of the α‐2,9 polysialyltransferase of Neisseria meningitidis

Abstract

Neisseria meningitidis Serogroup C is responsible for the majority of the meningococcal meningitis outbreaks in the United States in the past 20 years. The serogroup polysaccharide, an α‐2, 9 polysialic acid, of the pathogen is essential for virulence. This highlights the importance understanding of the enzymes involved in its biosynthesis as it may lead to the development of new drugs targeting this disease.We are interested in obtaining a detailed understanding of the kinetics of the α‐2, 9 polysialyltransferase involved in the formation of the polysaccharide capsule of N. meningitidis. Studies of the α‐2, 8 polysialyltransferase conducted by the Wakarchuk group obtained an apparent Km in the low mM range for CMP‐sialic acid. Using the α‐2, 9 polysialyltransferase we obtained an apparent Kmvalue in the low µM range for CMP‐sialic acid. We believe that this large discrepancy in Km is due primarily to the differences in assay methods and conditions.Using CMP‐sialic acid as the donor substrate, we compared acceptor substrate specificity of GD3, a ganglioside, with a hexamer of sialic acid. We observed a lower apparent Km value for GD3 compared with the hexamer suggesting that the attachment of a lipid to the oligosaccharide improves substrate binding. However, the enzyme does not appear to follow Michaelis‐Menten kinetics. The complexity of the reaction kinetics may be due to fact that each product formed via the transfer of sialic acid from CMP‐sialic acid in turn produces a new substrate. To address this issue we used CMP‐sialic acid analogs modified at the N‐9 position to observe a single turnover of product, which allowed us to perform detailed kinetic characterization of the enzyme.