The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyses the irreversible aldol-like condensation between the five-carbon phosphorylated aldose D-arabinose 5-phosphate (A5P), the three-carbon ester phosphoenolpyruvate (PEP) and water. This is the first committed step in the biosynthesis of 3-deoxy-D-manno-octulosonate (KDO), an essential lipopolysaccharide component of all Gram-negative bacteria and plants. KDO8PS is evolutionarily related to the enzyme 3-deoxy-D-arabino- heptulosonate 7-phosphate synthase (DAH7PS), which catalyses a similar aldol-like condensation concerning the four-carbon phosphorylated aldose D- erythrose 4-phosphate, PEP and water, in the first step of the shikimate pathway to aromatic compounds in microorganisms and plants. While the family of KDO8PS comprises both metal-independent and metal-dependent enzymes, all DAH7PS are metal-dependent enzymes.

DAH7PS can be categorised into type Iα, Iβ or II enzymes depending on what sequence additions are present, as these additional parts of the enzyme are also responsible for the feedback inhibition of DAH7PS. KDO8PS are suggested to have a common Iβ DAH7PS-like ancestor, with the metal-dependency lost later in time. KDO8PS can be categorised into metal-dependent bacterial, metal-independent bacterial and plant enzymes. All KDO8PS have the quaternary structure of a tetramer, with one plant KDO8PS also reported to be partially dimeric. The KDO8PS tetramer is comprised of the A-C tetramer interface and A-B tetramer interface. Most type Iβ DAH7PS have the quaternary structure of a tetramer, with only those with a C-terminal chorismate mutase domain able to form a unique dimer. The Iβ DAH7PS tetramer is comprised of the A-B tetramer interface and A-C tetramer interface. Little is understood about how role of the quaternary structure may change between metal-dependent bacterial, metal-independent bacterial and plant KDO8PS, and how it relates to the role of the quaternary structure in DAH7PS. This thesis describes the functional and structural studies of KDO8PS and DAH7PS, using the metal-independent bacterial KDO8PS from Neisseria meningitidis, the metal-dependent bacterial KDO8PS from Chlorobium tepidum the plant KDO8PS from Chlorela variabilis, and the type Iβ DAH7PS from Pyrococcus furiousus.

Chapter 2 investigates the role of a conserved Asp and Pro in the PEP binding pocket of KDO8PS. In DAH7PS the residues are conserved as a Glu and Gly. Mutational analysis found that in KDO8PS the Pro residue appears to be important for the formation of the A-C tetramer interface. Mutating this Asp and Pro in KDO8PS to Glu and Gly respectively was accommodated by KDO8PS but it was found the changes could potentially be optimised by coupling the change with mutation to other conserved differences.

The work described in Chapter 3 investigates the role of the quaternary structure in the metal-independent N. meningitidis KDO8PS. Individual residues of the A-C tetramer interface and A-B tetramer interface were mutated to destabilise the tetramer interfaces. It was found that the β2α2 loop interactions are not significant in the formation of the A-C tetramer interface. The β6α6 and β4α4 loop interactions were significant in the formation of the A-C tetramer interface, as mutation on these loops destabilised the A-C tetramer interface. The A-C tetramer interface was indicated to play an important role in enzyme catalysis and protein stability of metal-independent bacterial KDO8PS. The A-B tetramer interface was suggested to be essential for protein stability at the cost of greater catalytic activity in metal-independent bacterial KDO8PS, as mutation produced either insoluble protein or a more active tetramer.

The role of the quaternary structure in the type Iβ P. furiousus DAH7PS is probed in Chapter 4. Individual residues of the A-B tetramer interface and A- C tetramer interface were mutated to destabilise the tetramer interfaces. It was found that one mutation on the A-C tetramer interface destabilised both tetramer interfaces. As the DAH7PS monomer produced was catalytically inactive, the functional unit of DAH7PS is unlikely to be the monomer. The mutation identified that the β6α6 loop interactions were significant in the formation of the A-B tetramer interface. An A-B tetramer interface interaction between a Gln and Arg residue was indicated to be the source of E4P and PEP stabilisation with and without Cd2+ in DAH7PS.

In Chapter 5 a metal-dependent C. tepidum (Cte) KDO8PS and plant C. variabilis (Cvr) KDO8PS are identified for tetramer interface destabilisation experiments. The complete CteKDO8PS characterisation is achieved and is found to have similar properties to other characterised KDO8PS. CvrKDO8PS was found to have a unique five residue extension on the β6α6 loop not seen in any other KDO8PS sequence.

Lastly, in Chapter 6 all the chapter results are reviewed, and ideas are discussed for advancing the research presented in this thesis.