Heme is the oxygen-binding ligand of hemoglobin and is an essential cofactor or sensor element in many proteins. Heme production must be tightly controlled to adequately supply these functions but to avoid overproduction, as accumulation of free heme and heme precursors is toxic. The first committed step in heme biosynthesis is the condensation of glycine and succinyl-CoA to yield 5-aminolevulinic acid (ALA). This reaction is catalyzed by ALA synthase (ALAS), which uses pyridoxal 5ʹ-phosphate (PLP, the active form of vitamin B6) as an essential cofactor. In animals, there are two differentially expressed ALAS isoforms. ALAS1 is present in most cells, whereas ALAS2 is an erythroid-specific enzyme that is dramatically upregulated during red cell development. In humans, mutations in ALAS2 cause two diseases: (1) X-linked sideroblastic anemia (XLSA) when enzyme activity is too low to support healthy levels of heme production and erythropoiesis and (2) Erythroid X-linked protoporphyria (XLPP), from gain-of-function ALAS2 mutations that overproduce ALA, causing build up of toxic heme biosynthetic intermediates. The life cycle of ALAS is tightly regulated at steps including mitochondrial import and protein turnover. Both these steps are feedback controlled by heme-binding. Enzyme activity (and/or stability) is also regulated and these processes are affected by interaction with other enzymes, including Lon protease, succinyl-CoA synthetase (SCS), and perhaps ferrochelatase (FECH), the final two also critical enzymes in heme synthesis. Importantly, we recently discovered that ALAS activity is also dramatically stimulated by mitochondrial ClpX (mtClpX), a member of the AAA+ family of protein unfoldases. The mtClpX energy-dependent unfoldase accelerates incorporation of PLP into ALAS and CLPX depletion causes anemia in vertebrates. We also solved structures of both PLP-free ALAS (from yeast) and the active PLP-bound enzyme, which illuminates the conformational changes coupled to PLP incorporation and provides important information for understanding mtClpX-promoted loading of PLP. These structures also provide the first observation of the eukaryotic-specific regulatory C-terminal domain of the enzyme. This domain structure suggests testable mechanisms to explain the XLPP mutations and contains the binding site for SCS, which we will further study. Continuing to investigate how mtClpX physically interacts with ALAS and to test models for the mechanism of PLP-loading holds promise for uncovering a link between mtClpX-ALAS2 interactions and some classes of XLSA alleles. In another recent, exciting breakthrough, our collaborators discovered a dominant human CLPX mutation that appears to hyperactivate ALAS, leading to mtClpX-linked erythropoietic protoporphyria (EPP). The mechanistic basis of this disease will be scrutinized at the molecular, structural and cellular level. Thus, by probing the complex mechanisms that control ALAS enzymes we will elucidate new mol...