<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>
Structural Basis for Competitive Inhibition of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase fromVibrio cholerae
