Substrate-bound structures of yeast ALA synthase reveal protein dynamics initiating heme biosynthesis.

Heme is an essential prosthetic group involved in multiple biological processes in nearly all organisms. Heme biosynthesis is initiated by the pyridoxal 5'-phosphate-dependent enzyme aminolevulinic acid synthase (ALAS). Mutations in ALAS cause multiple hematological diseases. Despite sharing approximately 70% sequence similarity, ALAS homologs exhibit distinct expression and regulatory mechanisms. However, the molecular basis for their functional divergence remains poorly understood. Here, we used x-ray crystallography and molecular dynamics simulations to elucidate the structure and dynamics of Saccharomyces cerevisiae ALAS (scALAS) upon substrate binding. We identified critical conformational changes that occur during catalysis, regulating enzyme activity and protein-protein interactions specific to eukaryotic heme synthesis. Using evolution-guided engineering, we generated scALAS variants with altered catalytic activity and cellular metabolic profiles. Our findings reveal molecular and structural mechanisms underlying heme synthesis regulation and establish a framework for developing targeted therapeutics to treat heme-deficiency disorders and inform a rational design of next-generation therapeutic tools.