Structural investigation of ALA synthase reveals allosteric regulation governed by protein dynamics

Abstract

Pyridoxal 5’-phosphate (PLP), a bioactive form of Vitamin B6, serves as a cofactor for over 140 distinct enzyme activities that are essential to biological survival. The reactivity of PLP places responsibility on the enzyme scaffold to mediate reaction type, therefore PLP-dependent enzymes (PDEs) can be categorized into fold types based on structural conservation. The differences lie in their distinct, allosteric mechanisms that help to regulate enzyme function. Since allostery relies on the interconversion of conformational states, understanding the protein dynamics within a system will provide an opportunity to target specific PDEs for therapeutic and industrial use. In this dissertation, I have used an integrated approach involving structural, computational, biophysical, and biochemical techniques to explore the autoregulation of a member of the PDE Fold Type I superfamily. Aminolevulinic acid synthase (ALAS), a heme biosynthetic enzyme, contains a conserved, catalytic core, but eukaryotic homologs have evolved to include divergent, extended termini that differentially interact with the core. As a result, mutations spanning across conserved and divergent regions result in opposite functional phenotypes. My work demonstrates that the basis of these differences lies in the complexity and interconnectivity of eukaryotic ALAS protein dynamics. Unlike bacterial ALAS dynamics which rely solely on loop movement proximal to the active site, eukaryotic dynamics communicate across a wider region, encompassing both the core and divergent termini. This wide network of intra-molecular contacts further modulates inter-molecular interactions with ligands and other proteins, meaning that ALAS homologs have each adapted to function in their respective cellular contexts by fine-tuning protein dynamics. Structural and bioinformatic analysis of Fold Type I PDEs reveals similar conformational evolution, demonstrating that this case study of ALAS provides a foundation for the investigation of precise allosteric mechanisms for this family of enzymes.