Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Abstract

Homocitrate synthase (HCS) catalyzes the first and committed step in the α-aminoadipate (AAA) pathway of lysine biosynthesis, which occurs in many fungi and certain archaea, and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is comprised of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed α/β-topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain covers the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex, the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Steady state kinetic assays and in vivo yeast growth assays on wild-type enzyme and active site mutants allow us to elucidate its catalytic mechanism, including the residues implicated in catalysis. Together these results yield new insights into the mechanism and regulation of HCS, which provide a platform to identify small molecule inhibitors of HCS that may be optimized and used as anti-fungal agents.