The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway leading to the synthesis of aromatic amino acids. The pathway is unique to microorganisms and plants. Feedback regulation of DAH7PS activity is important for regulating the supply of pathway end-products.¹

All DAH7PSs share a basic catalytic (β/α)₈-barrel, which is often decorated with additional structural elements. These extensions vary in their nature and are implicated in the allosteric regulation of DAH7PS activity. Analysis of the type Iβ DAH7PS subfamily indicates the presence of an N-terminal extension (Geobacillus sp.) attached to the catalytic barrel with sequence similarity to chorismate mutase (CM).¹ Similarly, the catalytic core of the type Iβ DAH7PS from Thermotoga maritima bears an N-terminal ACT domain.²  Both these N-terminal domains are responsible for an allosteric response. In contrast, the DAH7PS from Pyrococcus furiosus (PfuDAH7PS) bears no regulatory machinery and hence is unregulated.¹

In these studies we have constructed protein chimera to mix and match catalytic and regulatory domains in order to add or swap allosteric functionality. These studies provide insight into the modular evolution of allostery and illustrate the ease with which functional allostery can be acquired by gene fusion events.

Structure and amino acid sequence alignments of the catalytic DAH7PS domains from PfuDAH7PS, GspDAH7PS, and TmaDAH7PS show high level of conservation of the key residues involved in regulation.  P. furiosus expresses a separate CM (PfuCM) and the genes encoding PfuDAH7PS and PfuCM are located independently in the genome. By design and construction of three fusion proteins, PfuCM-PfuDAH7PS, GspCM-PfuDAH7PS, GspCM-TmaDAH7PS, our studies aim to introduce allosteric regulation to PfuDAH7PS by addition of a putative regulatory domain, as well as exchange regulation between GspDAH7PS and TmaDAH7PS.