Download MendeleyThree-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism.
Schmidt, A., Sivaraman, J., Li, Y., Larocque, R., Barbosa, J.A.R.G., Smith, C., Matte, A., Schrag, J.D., Cygler, M.(2001) Biochemistry 40: 5151-5160
- PubMed: 11318637
- DOI: https://doi.org/10.1021/bi002204y
- Primary Citation of Related Structures:
1FC4 - PubMed Abstract:
2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.
Organizational Affiliation:
Biotechnology Research Institute and Montr¨¦al Joint Centre for Structural Biology, National Research Council of Canada, Montr¨¦al, Qu¨¦bec H4P 2R2, Canada.
