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. 2018 Jul 16;11(1):482.
doi: 10.1186/s13104-018-3596-y.

Determination of the binding affinities of Neisseria meningitidis serogroup W capsule polymerase with two nucleotide sugar substrates

Abeer Sharyan  1 , Cendy Gonzalez  2 , Ophelia Ukaegbu  1 , Kayla Powell  1 , Pumtiwitt C McCarthy  3
Affiliations

Determination of the binding affinities of Neisseria meningitidis serogroup W capsule polymerase with two nucleotide sugar substrates

Abeer Sharyan et al. BMC Res Notes. .

Abstract

Objective: Meningococcal meningitis is a public health burden. Immunization strategies have reduced global incidence of the disease. Glycoconjugate vaccines are the most effective type of vaccine to combat most causes of meningococcal meningitis. These vaccines contain capsular polysaccharide fragments from disease-causing serogroups of Neisseria meningitidis that are chemically attached to a carrier protein. The enzymes responsible for capsular polysaccharide synthesis can serve as tools to make these critical vaccine components. One such enzyme is the N. meningitidis serogroup W capsule polymerase. This enzyme is responsible for creating the galactose-sialic acid containing capsular polysaccharide of this serogroup. Our aim in this study was to determine the binding affinities of nucleotide sugar donors CMP-sialic acid and UDP-galactose using a coupled transferase assay to inform future work to modulate polysaccharide synthesis by this enzyme.

Results: We determined a Km of 66.8 µM for CMP-sialic acid and a Km for UDP-galactose of 3.9 µM. These values are lower than reported values for other retaining galactosyltransferases and inverting sialyltransferases respectively. There were difficulties obtaining reliable data for galactosyltransferase activity. An alternate strategy is needed to assess kinetic parameters of the separate transferase activities for this enzyme.

Keywords: Enzyme kinetics; Glycosyltransferases; Neisseria meningitidis; Vaccine development.

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Figures

Fig. 1
Fig. 1
Schematic of the multi-enzyme coupled transferase assay. UDP-galactosyltransferase activity of the serogroup W capsule polymerase can be measured separately from sialyltransferase activity. The boxed reactions indicate reactions that take place in the presence of NMPK
Fig. 2
Fig. 2
Kinetic effects of varying UDP-galactose concentrations on UDP-galactosyltransferase activity of the N. meningitidis serogroup W capsule polymerase. Reactions were performed in triplicate. The mean is plotted, and error bars represent standard deviation. a Michaelis–Menten plot and b Lineweaver–Burke plot
Fig. 3
Fig. 3
Kinetic effects of varying CMP-sialic acid concentrations on activity of the N. meningitidis serogroup W capsule polymerase. Reactions were performed in triplicate. The mean is plotted, and error bars represent standard deviation. a Michaelis–Menten plot and b Lineweaver–Burke plot
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