This proposal seeks to investigate catalysis of a capsule polymerase enzyme from Neisseria meningitidis, one of the leading bacterial causes of meningitis. This capsule around the bacteria is rich in polysaccharides. Serogroup-specific enzymes are responsible for synthesis of these carbohydrates. The capsule enzymes that synthesize polymers of a single sugar have been well studied compared to those that synthesize polymers containing two different sugars. As a result, much less is known about catalysis by these heteropolymeric capsule polymerase enzymes. This class of enzymes provides one target for vaccine research and drug development. The overall goal of this work is to develop an understanding of the Neisseria meningitidis serogroup W capsule polymerase enzyme which makes a polysaccharide containing alternate repeats of two different sugars. Our goal in this work is to perform series of fundamental experiments that will provide insight into this enzyme's mechanism. This goal will be accomplished by the following specific aims:
Aim 1 : Determine the kinetics of catalysis by the N. meningitidis serogroup W capsule polymerase. We will perform kinetics measurements using an HPLC-based fluorescent assay. To carry out this assay, we will first define optimal fluorescent acceptors. The knowledge gained from completion of this aim (kinetic parameters [Km, Vmax, kcat] of the enzyme that are currently unknown), will provide important insight into catalytic rates and mechanism.
Aim 2 : Determine key amino acids critical to N. meningitidis serogroup W capsule polymerase catalysis. We will chemoenzymatically synthesize a photoactivatable substrate analog, CMP-SiaDAz, which will be used to achieve photoactivated labeling of binding site residues. We will determine the location of modified amino acids by mass spectrometry analysis and perform activity assay of mutant forms of the protein. The knowledge gained will provide new insight into substrate recognition and binding during enzyme catalysis.
Aim 3 : Use chain-terminating nucleotide sugar donors to rationally control polysaccharide synthesis. We will react modified nucleotide donor sugars with the enzyme under various conditions and assess polysaccharide composition/chain length. Completion of this aim will reveal how this enzyme can react with alternate substrates leading to new potential enzyme inhibitors.
Neisseria meningitidis poses a global threat to developed and undeveloped nations across the world. This proposal seeks to probe the molecular mechanism of an enzyme that produces an essential carbohydrate polymer on the outer surface of the bacteria. Gaining this mechanistic knowledge will aid in the development of new, targeted therapies against N. meningitidis.
