# Mechanistic Studies of a Bacterial Capsule Polymerase

> **NIH NIH SC2** · MORGAN STATE UNIVERSITY · 2020 · $151,000

## Abstract

PROJECT SUMMARY
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.

## Key facts

- **NIH application ID:** 9942481
- **Project number:** 5SC2GM125517-03
- **Recipient organization:** MORGAN STATE UNIVERSITY
- **Principal Investigator:** Pumtiwitt McCarthy
- **Activity code:** SC2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $151,000
- **Award type:** 5
- **Project period:** 2018-07-26 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9942481

## Citation

> US National Institutes of Health, RePORTER application 9942481, Mechanistic Studies of a Bacterial Capsule Polymerase (5SC2GM125517-03). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9942481. Licensed CC0.

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