Neisseria Meningitidis Serogroup W Capsule Polymerase: Determining Enzyme Kinetics Of Donor Sugars And Development Of A Chemoenzymatic Method To Produce A Photo-Crosslinking Derivative
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Type of WorkText
ProgramMaster of Science
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Most cases of bacterial meningitis are caused by one of three bacteria, Streptococcus pneumonia, Haemophilus influenza or Neisseria meningitidis. The overall aim of our work is to better understand the molecular mechanism of Neisseria meningitidis serogroup W capsule polymerase enzyme. The capsule that surrounds the outer surface of the bacteria is carbohydrate-rich. Capsular polysaccharides help protect the bacteria against the human immune system.The serogroup W capsule contains sialic acid-galactose heteropolymers and uses CMP-sialic acid and UDP-galactose as sugar donors during synthesis. Our long-term goal is to understand how this enzyme performs catalysis. This knowledge can provide new insight for drug and vaccine development against N. meningitidis. One goal of this thesis work was to determine the kinetic parameters (Km, Vmax, kcat) for each nucleotide donor sugar with the recombinant protein. An absorbance-based multi-enzyme method was used in which activity of the N. meningitidis serogroup W enzyme was linked to NADH oxidation. Kinetic parameters were established for both donors, however initial work suggests further testing is required for CMP-sialic acid. The second goal of this work was to develop a chemoenzymatic method to produce a photo-crosslinking derivative of CMP-sialic acid (diazirine modified, CMP-SiaDAz). A strategy, using two recombinant enzymes from the sialic acid biosynthetic pathway, was used first to synthesize CMP-Sialic acid. Once success was confirmed this same system was used to synthesize CMP-SiaDAz. Light-activated crosslinking of the product with N. meningitidis serogroup W protein was performed. Afterwards the serogroup W capsule polymerase was incubated with endoproteinase and peptide fragments were analyzed by SDS-PAGE gel electrophoresis and mass spectrometry. Endoproteinase digestion was successful; however crosslinking analysis was inconclusive. This thesis work is an important step towards understanding catalysis by the N. meningitidis serogroup W capsule and future work will focus on optimizing current methods and repeating experiments.