Structure, Mechanism and Regulation of Homocitrate Synthase.

Homocitrate Synthase;Amino Acid Biosynthesis;Structural Biology;Biological Chemistry;Science;Biological Chemistry

Bulfer, Stacie LynneSmith, Janet ;

University of Michigan

关键词: Homocitrate Synthase;    Amino Acid Biosynthesis;    Structural Biology;    Biological Chemistry;    Science;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/76021/sbulfer_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The amino acid lysine is synthesized via two distinct pathways with the alpha-aminoadipate (AAA) pathway being utilized by fungi and certain archaebacteria. The first and committed step of this pathway is catalyzed by homocitrate synthase (HCS), which transfers an acetyl group from acetyl-Coenzyme A to 2-oxoglutarate (2-OG) to yield homocitrate and Coenzyme A. Additionally, HCS plays a role in the regulation of the AAA pathway, as it is feedback inhibited by the end product, L-lysine. Due to the absence of HCS in humans and its importance in the AAA pathway, this enzyme represents a potential target for small molecule inhibitors that may be used as broad-spectrum antifungal therapies to treat fungal infections, which occur frequently in immuno-compromised individuals. To better understand the mechanism of HCS, we determined the first crystal structure of a fungal HCS from Schizosaccharomyces pombe, along with two different structures of enzyme in complex with 2-OG. The structure reveals that 2-OG binds in the active site within the (alpha/beta)8 TIM barrel domain via hydrogen bonds and coordination to the active site divalent metal ion. Steady state kinetic analysis and in vivo growth studies of mutations of residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity revealing the roles of these amino acids in substrate binding or the catalytic mechanism of HCS. Similar studies were completed to elucidate the mechanism of feedback regulation of HCS by L-lysine. The structure of an HCS/L-lysine complex illustrates that the inhibitor binds in the same binding site as the substrate 2-OG. In vitro and in vivo assays on mutants that interact with L-lysine impair feedback inhibition, as do previously identified substitutions of residues outside of the active site that render HCS insensitive to L-lysine. Finally, we utilized a high-throughput screening (HTS) approach to identify small molecule inhibitors of HCS. Collectively, these structural and functional studies elucidate the catalytic and regulatory mechanisms of HCS. Furthermore, they provide a framework for discovery of inhibitors to HCS either through HTS or structure-based design, which may be developed into drugs to treat fungal infections.