Neisseria meningitidis is a Gram-negative bacterium that is responsible for most cases of bacterial meningitis. Our long-term goal seeks to understand catalysis by the N. meningitidis serogroup W capsule polymerase. This enzyme could provide an important tool for synthesis of polysaccharides for vaccine development. This glycosyltransferase synthesizes a heteropolysaccharide of galactose and sialic acid that surrounds the outer surface of the bacteria. A suitable assay is critical to investigating kinetics and binding properties of this capsule polymerase. Currently we are optimizing two assays to test activity of this enzyme. The first is a fluorescence-based method using fluorescent sugar acceptors in an HPLC based assay. Non-fluorescent sugars were chemically modified to include fluorescent dyes either by click 1,3 cycloaddition chemistry, chemoenzymatic synthesis, or through direct incorporation. The second is a multi-enzyme absorbance-based method. This method links the N. meningitidis serogroup W polymerase activity to NADH oxidation that can be followed spectrophotometrically. Initial work suggests that both assays will be useful in studying this enzyme however further optimization is needed. Future work will continue optimization.

Biochemical assay development for a Neisseria meningitidis capsule polymerase

254th National Meeting (2017)

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