Acyl group specificity at the active site of tetrahydridipicolinate N-succinyltransferase

Abstract

Tetrahydrodipicolinate N-succinyltransferase (DapD) catalyzes the succinyl-CoA-dependent acylation of L-2-amino-6-oxopimelate to 2-N-succinyl-6-oxopimelate as part of the succinylase branch of the meso-diaminopimelate/lysine biosynthetic pathway of bacteria, blue-green algae, and plants. This pathway provides meso-diaminopimelate as a building block for cell wall peptidoglycan in most bacteria, and is regarded as a target pathway for antibacterial agents. We have solved the X-ray crystal structures of DapD in ternary complexes with pimelate/succinyl-CoA and L-2-aminopimelate with the nonreactive cofactor analog, succinamide-CoA. These structures define the binding conformation of the cofactor succinyl group and its interactions with the enzyme and place its thioester carbonyl carbon in close proximity to the nucleophilic 2-amino group of the acceptor, in support of a direct attack ternary complex mechanism. The acyl group specificity differences between homologous tetrahydrodipicolinate N-acetyl- and N-succinyltransferases can be rationalized with reference to at least three amino acids that interact with or give accessible active site volume to the cofactor succinyl group. These residues account at least in part for the substrate specificity that commits metabolic intermediates to either the succinylase or acetylase branches of the meso-diaminopimelate/lysine biosynthetic pathway.

Fig. 1.

(A) The reaction sequence leading from tetrahydrodipicolinate to lysine via the succinylase branch of the meso-diaminopimelate/lysine biosynthetic pathway. (B) The substrates and analogs used in this study.

Fig. 2.

Overall structure of trimeric DapD in complex with pimelate (white) and succinyl-CoA (yellow). Each trimer contains three active sites. (A) View parallel to the molecular threefold axis. (B) View perpendicular to the threefold axis. The proximal active site is composed of two subunits termed A (green) and B (blue).

Fig. 3.

Succinyl group binding pocket of DapD. (A) Stereoview of the unbiased Fo-Fo electron density map surrounding the refined coordinates corresponding to the succinamide moiety of the succinamide-CoA cofactor analog. (B) Hydrophilic interactions at the active site of the pimelate/succinyl-CoA complex. (C) Hydrophilic interactions at the active site of the L-2-aminopimelate/succinamide-CoA complex. (D) Superimposed stereoview of the two complexes, depicting the acceptor and cofactor (or analogs), surrounding water molecules, and the two residues that interact with the succinyl carboxyl group (Arg 187A, Glu 189B). The nucleophilic amino group of L-2-aminopimelate and the carbonyl carbon of the succinyl-CoA thioester are joined by a dotted line (distance 2.9 Å).