N-Acetylneuraminic acid synthase (NANAS) is an enzyme responsible for the biosynthesis of N-acetylneuraminic acid (NANA). NANA is the most common form of a group of nine carbon sugar molecules called the sialic acids. NANA production is common in mammalian cells for vital physiological processes. A few species of microorganisms, including pathogenic bacteria such as Neisseria meningitidis and Campylobacter jejuni, are known to synthesise NANA by their bacterial NANAS. These pathogenic bacteria synthesise NANA for molecular mimicry, allowing them to evade the host immune system. This thesis examines the NANAS enzymes from N. meningitidis and C. jejuni. Inhibitory studies with these enzymes were explored by performing enzyme kinetics with substrate analogues and a product analogue which structurally mimic the natural substrates or product of NANAS. Inhibition constants were determined for a variety of analogues to give insight in to how the enzyme accommodates its substrates within the active site of NANAS. This study may be a useful step in the development of alternative antibiotics for bacterial meningitis and other diseases in the future. NANAS catalyses a condensation reaction between phosphoenolpyruvate (PEP) and N-acetyl mannosamine (ManNAc). Both PEP and ManNAc analogues were explored as inhibitors of the enzymes. Results from this study show that increasing steric bulk of the substituents at C3 of PEP unexpectedly delivers more potent inhibition of the enzyme. This finding suggests that a slightly modified binding position of the PEP analogue within the PEP binding site of the enzyme may be responsible for the inhibition. A reduced acyclic analogue of ManNAc was found to be an effective inhibitor of the enzymes. This finding indicates how important the acyclic form of ManNAc is in the reaction mechanism catalysed by this enzyme.

Sialic acids are prevalent in many organisms and facilitate a range of cellular processes in both bacteria and mammals. Whilst a variety of sialic acids are present in nature, N-Acetylneuraminic acid (NANA) is the most common and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host’s immune system. NANA is synthesised in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalysed by the domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). Each NANAS monomer is comprised of two distinct domains; a catalytic domain linked to an antifreeze protein-like (AFPL) domain. This thesis outlines research into the role of the AFPL domain using a range of structural and kinetic analyses to compare variant enzymes to the natural, NmeNANAS enzyme. An investigation was also made into the evolutionary relationships between NANAS and other bacterial sialic acid synthases such as Legionaminic acid synthase and Pseudaminic acid synthase.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Similar to other non-enveloped viruses, the mechanism of cell entry for polyomaviruses is poorly understood. The polyomavirus capsid is an icosahedron composed of 72 pentamers of the major capsid protein VP1. There is one copy of minor capsid proteins, VP2 or VP3, at the center of each pentamer. According to previous studies, polyomavirus cell entry is a multi-step process which includes: 1) VP1 binding to sialic acid (SA) on the surface of host cells, 2) interaction of VP1 with alpha4beta1 integrin and 3) subsequent cell penetration. Biochemical studies have shown that SA alters polyomavirus protease sensitivity, suggesting a conformational change. The aim of this study was to determine these conformational changes at the molecular level. Therefore, we used single particle cryo-electron microscopy to construct 3D maps of wild type (WT) murine polyomavirus, WT bound to SA, a mutant with a disrupted integrin binding site, and the mutant bound to SA. Our results reveal that in both WT and mutant viruses, a significant conformational change happens after binding with SA which is seen as an additional ring of density inside the virus. Moreover some negative densities are seen in the difference map of WT and WT bound with SA, which suggests movement of some viral proteins after binding with SA.

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