Linker Dynamics in Allosteric Regulation of 3-Deoxy-D-arabino-heptulosonate 7-phosphate Synthase (2016)
AuthorsMoore, Jordyn Alissashow all
The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step in the shikimate pathway, the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P); leading to production of the aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr). DAH7PS enzymes are classified into two homologous families, type I and type II, differentiated by molecular weight and amino acid sequence. The type I DAH7PS enzymes are further sub-divided into type Iα and type Iβ, based on amino acid sequence. DAH7PS enzymes share a common TIM barrel fold, which comprises the key catalytic residues required for DAH7PS activity. The presence of recruited structural elements to the core TIM barrel fold, confers allosteric regulation to the DAH7PS enzymes. The aim of this thesis is to explore the role of linker dynamics in allosteric regulation of the type Iβ DAH7PS enzyme, from the bacterium Thermotoga maritima. The hypothesis that the inter-domain linker determines molecular motion of the regulatory domains and consequently is responsible for allosteric regulation of the enzyme will be investigated. Chapter two describes the functional and structural characterisation of T. maritima DAH7PS (TmaDAH7PS) and three TmaDAH7PS linker variants, each involving a single amino acid substitution within the linker sequence. The response of the TmaDAH7PS linker variants to inhibitors will be investigated and compared to the wild-type TmaDAH7PS enzyme (WTTmaDAH7PS). Chapter three considers the role of the inter-domain linker in molecular motion of the enzyme, linking protein dynamics to the allosteric regulation mechanism employed by TmaDAH7PS. The studies presented in this chapter focus on the V65PTmaDAH7PS linker variant, where a valine (Val) at position 65 has been substituted for proline (Pro). Comparisons to WTTmaDAH7PS are drawn. Chapter four combines the results of chapters two and three to present a more comprehensive discussion of the role of linker dynamics in the allosteric regulation mechanism employed by TmaDAH7PS. Chapter five provides a summary of the work presented in this thesis, considering future work that would further understanding of the role of linker dynamics in TmaDAH7PS allostery.