Destabilization of the Homotetrameric Assembly of 3-Deoxy-d-Arabino-Heptulosonate-7-Phosphate Synthase from the Hyperthermophile Pyrococcus furiosus Enhances Enzymatic Activity

Abstract

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAH7PS), adopts a variety of different quaternary structures depending on the organism in which it is found. The DAH7PS from the hyperthermophilic archaebacterium Pyrococcus furiosus was previously shown to be tetrameric in its crystalline form, and this quaternary association is confirmed in an improved structure in a different crystal system. This tetramer is also present in solution as revealed by small-angle X-ray scattering and analytical ultracentrifugation. This homotetrameric form has two distinct interfaces, both of which bury over 10% each of the surface area of a single monomer. Substitution of Ile for Asp in the hydrophobic region of one interface gives a protein with a remarkable 4-fold higher maximum catalytic rate than the wild-type enzyme. Analytical ultracentrifugation at pH7.5 reveals that the tetrameric form is destabilized; although the protein crystallizes as a tetramer, equilibrium exists between tetrameric and dimeric forms with a dissociation constant of 22μM. Thus, under the conditions of kinetic assay, the enzyme is primarily dimeric, revealing that the dimeric form is a fully functional catalyst. However, in comparison to the wild-type protein, the thermal stability of the dimeric protein is significantly compromised. Thus, an unusual compromise of enzymatic activity versus stability is observed for this DAH7PS from an organism that favors a hyperthermophilic environment.