Three-dimensional structure of 2-amino-3-kelobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: Inferred reactionmechanism

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phospha te (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with t hreonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of c ofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escheri chia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 Angstrom resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspart ate aminotransferase. Its closest structural homologue is E. coli 8-amino-7 -oxononanoate synthase. Like many other members of the ct family, the funct ional 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/ subst rate binding site. Electron density maps indicated the presence in the crys tal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an e xternal aldimine, which remained bound to KBL throughout the protein purifi cation procedure. The observed interactions between the aldimine and the si de chains in the substrate binding site explain the specificity for the sub strate and provide the basis for a detailed proposal of the reaction mechan ism of KBL. A putative binding site of the CoA cofactor was assigned, and i mplications for the cooperation with threonine dehydrogenase were considere d.