Three-Dimensional Structure of 2-Amino-3-ketobutyrate CoA Ligase from Escherichia coli Complexed with a PLP-Substrate Intermediate: Inferred Reaction Mechanism Andrea Schmidt, J. Sivaraman, Yunge Li, Robert Larocque, Joa ˜o A. R. G. Barbosa, Christopher Smith, Allan Matte, Joseph D. Schrag, and Miroslaw Cygler* Biotechnology Research Institute and Montre ´ al Joint Centre for Structural Biology, National Research Council of Canada, 6100 Royalmount AVenue, Montre ´ al, Que ´ bec H4P 2R2, Canada ReceiVed September 19, 2000; ReVised Manuscript ReceiVed February 23, 2001 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 Å resolution. KBL belongs to the R 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 R 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. Threonine degradation occurs via three pathways; the main pathway involves a two-step process that converts threonine to glycine (1-3). This pathway (Figure 1) is common to prokaryotic and eukaryotic cells and involves two enzymes: L-threonine dehydrogenase [TDH, 1 EC 1.1.1.103 (4-6)] and 2-amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) (2, 3, 7-9). In the first step, which requires the cofactor NAD, TDH converts L-threonine into L-2-amino-3-ketobutyrate. This intermediate then serves as a substrate for KBL, which, in the presence of CoA, catalyzes the conversion of 2-amino- 3-ketobutyrate to glycine and acetyl-CoA. Glycine can be utilized as an educt for serine synthesis, while acetyl-CoA is utilized in a large variety of metabolic reactions [e.g., EcoCyc (10)]. The reactions catalyzed by TDH and KBL are coupled, both in vivo and in vitro (9), and the two enzymes have been shown to form a complex, most likely containing one tetramer of TDH and two dimers of KBL (7, 8, 11). This concerted mode of action is consistent with the highly reactive nature of the 2-amino-3-ketobutyrate inter- mediate, which in aqueous solution undergoes spontaneous decarboxylation to yield aminoacetone and CO 2 . The equi- librium between threonine, glycine, 2-amino-3-ketobutyrate, and aminoacetone is determined by the intracellular con- centrations of threonine, glycine, NAD, and CoA (12). * Corresponding author. Tel: (514) 496-6321. Fax: (514) 496-5143. E-mail: mirek.cygler@bri.nrc.ca. 1 Abbreviations: CoA, coenzyme A; NAD, nicotinamide adenine dinucleotide; KBL, 2-amino-3-ketobutyrate CoA ligase; PLP, pyridoxal 5′-phosphate; TDH, L-threonine dehydrogenase; AONS, 8-amino-7- oxononanoate synthase; AON, 8-amino-7-oxononanoate. FIGURE 1: Threonine degradation (or synthesis) pathway via threonine dehydrogenase (TDH) and 2-amino-3-ketobutyrate CoA ligase (KBL). Without KBL, the main product of threonine oxidation would be aminoacetone. The equilibrium between all of these compounds is determined mainly by the relative amounts of threonine, NAD, glycine, and CoA. Figure produced in Chem- Windows 5.0 (SoftShell Int. Ltd.)/CorelDraw 7. 5151 Biochemistry 2001, 40, 5151-5160 10.1021/bi002204y CCC: $20.00 © 2001 American Chemical Society Published on Web 04/05/2001