ABSTRACT: Heme is a critical biomolecule that carries out several functions in nearly all life forms, including humans where its most widely known role is mediating oxygen transport in the blood. It is imperative that heme production is tightly controlled as alterations in cellular heme levels can have drastic consequences for human health. The first and rate-limiting enzyme controlling heme biosynthesis is aminolevulinic acid synthase (ALAS). ALAS is conserved in α- proteobacteria and non-plant eukaryotes, however, an important structural feature found exclusively in eukaryotic ALAS enzymes is the C-terminal extension. We seek to understand how ALAS interacts with accessory proteins as well as organism-specific differences in protein assembly that may alter the regulation of heme production. Therefore, we use X-ray crystallography combined with biophysical and biochemical characterization of various eukaryotic ALAS enzymes to parse apart the role of this key regulatory region in eukaryote ALAS function. Our work is beginning to reveal key structure-function relationships between the orientation and molecular contacts mediated by the C-terminus and ALAS enzyme function, thus controlling heme biosynthesis.