Structural basis for regulation in gram-negative bacterial citrate synthases.

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Citrate synthases in Gram-negative bacteria differ from eukaryotic forms, notably in allosteric NADH inhibition.
• Despite functional differences, bacterial and eukaryotic citrate synthases share ~30% amino acid sequence homology.

Purpose of the Study:

• To investigate the allosteric NADH inhibition mechanism in Escherichia coli citrate synthase.
• To characterize the binding sites and regulatory roles of NADH and 2-oxoglutarate.

Main Methods:

• Construction of a structural model for E. coli citrate synthase based on sequence homology and pig heart enzyme structure.
• Identification of key cysteine residues (e.g., Cys-206) potentially involved in NADH binding.
• Generation and analysis of E. coli citrate synthase mutants (deletion and missense) using oligonucleotide-directed mutagenesis.

Main Results:

• The structural model suggests the identified reactive cysteine (Cys-206) is distant from the active site, supporting an allosteric NADH binding site.
• A deletion mutant lacking 24 amino acids near the active site retained normal NADH binding.
• Active site mutants exhibited reduced oxaloacetate affinity and significantly decreased sensitivity to 2-oxoglutarate.

Conclusions:

• NADH binds to a distinct allosteric site in E. coli citrate synthase, separate from the active site.
• 2-oxoglutarate acts as an active-site directed inhibitor, not an allosteric one, though it may retain in vivo regulatory functions.