Crystal structure and functional analysis of homocitrate synthase, an essential enzyme in lysine biosynthesis.
Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis 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 composed 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 alpha/beta topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes 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. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.
Bulfer
Stacie L
SL
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Scott
Erin M
EM
Couture
Jean-François
JF
Pillus
Lorraine
L
Trievel
Raymond C
RC
D020134:Catalytic Domain / Q000502:physiology; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007656:Ketoglutaric Acids / Q000737:chemistry* / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D007656:Ketoglutaric Acids; D029702:Schizosaccharomyces pombe Proteins; C021365:homocitrate synthase; D007652:Oxo-Acid-Lyases; D008239:Lysine