Optimizing a Chemoenzymatic Method to Produce a Photocrosslinking Nucleotide Donor Sugar for the N. meningitidis Serogroup W Capsule Polymerase
Authors: Gonzalez Cendy 
Published: November 2023

Most cases of bacterial meningitis are caused by one of three bacteria, Streptococcus pneumonia, Haemophilus influenza or Neisseria meningitidis. Bacterial meningitis affects about one fifth of the human population. Our work focuses on one type of bacteria that causes the disease, Neisseria meningitidis serogroup W. The capsule that surrounds the bacteria is carbohydrate‐rich. Polysaccharides in the capsule help protect the bacteria against the human immune system. This serogroup contains sialic acid‐galactose heteropolymers. The serogroup W capsule polymerase enzyme uses CMP‐sialic acid and UDP‐galactose as nucleotide donor sugars during synthesis of the heteropolymer. Our overall goal is to better understand the molecular mechanism of this enzyme. To help us achieve this goal, we are developing a chemoenzymatic method to produce modified CMP‐sialic acid diazirine derivatives with photo crosslinking ability. These derivatives will be used to label potential enzyme active site residues. Recombinant forms of Camplyobacter jejuni sialic acid synthase and Neisseria meningitidis CMP‐sialic acid synthase were used. Sialic acid synthase produces sialic acid from N‐acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). CMP‐sialic acid synthetase uses cytidine 5′‐triphosphate disodium salt (CTP) and sialic acid to synthesize product. Both enzymes were expressed in E. coli and a purification strategy for CMP‐sialic acid synthase using nickel affinity chromatography was developed. The sialic acid synthase was used as a crude lysate. Enzyme activity was tested in a trial one pot chemoenzymatic synthesis. Four separate samples were prepared containing ManNAc, PEP, CTP and a fluorescent acceptor in the presence or absence of enzymes. These enzymes were sialic acid synthase, CMP sialic acid synthase, and Camplyobacter jejuni CST II. For all three control samples, only one enzyme was added and the remaining two were missing. The remaining sample served as our reaction and contained all three enzymes. The results were visualized by thin layer chromatography (TLC) and indicated that enzymes CMP‐sialic acid was successfully made and CSTII was able to use this to sialylate a fluorescent acceptor. These results led us to perform a similar one pot chemoenzymatic synthesis conducted with diazirine‐modified N‐acetylmannosamine (ManNDAz) to make modified CMP‐sialic acid (CMP‐SiaDAz). These reactions did not contain CST II or acceptor. Three samples were used; two controls contained ManNDAz, PEP, CTP and either sialic acid synthase or CMP‐sialic acid synthetase. The reaction sample contained both. These samples were tested by TLC and p‐anisaldehyde sugar staining. Results from all three samples were compared to results from TLC of individual samples of PEP, ManNAc, sialic acid, CMP‐sialic acid and ManNDAz. These preliminary results proved inconclusive. Future work will continue to develop our chemoenzymatic method for modified CMP‐sialic acid (CMP‐SiaDAz) and optimize qualitative and quantitative detection of products formed.