Three-Dimensional Structure of Escherichia coli Dihydrodipicolinate Reductase in Complex with NADH and the Inhibitor 2,6-Pyridinedicarboxylate^,

Abstract

Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the alpha,beta-unsaturated cyclic imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicolinate. The enzyme is a component of the biosynthetic pathway that leads to diaminopimelate and lysine in bacteria and higher plants. Because these pathways are unique to microorganisms and plants, they may represent attractive targets for new antimicrobial or herbicidal compounds. The three-dimensional structure of the ternary complex of Escherichia coli dihydrodipicolinate reductase with NADH and the inhibitor 2,6-pyridinedicarboxylate has been solved using a combination of molecular replacement and noncrystallographic symmetry averaging procedures and refined against 2.6 A resolution data to a crystallographic R-factor of 21.4% (Rfree is 29.7%). The native enzyme is a 120 000 molecular weight tetramer of identical subunits. The refined crystallographic model contains a tetramer, three molecules of NADH, three molecules of inhibitor, one phosphate ion, and 186 water molecules per asymmetric unit. Each subunit consists of two domains connected by two flexible hinge regions. While three of the four subunits of the tetramer have a closed conformation, in which the nicotinamide ring of the cofactor bound to the N-terminal domain and the reducible carbon of the substrate bound to the substrate binding domain are about 3.5 A away, the fourth subunit is unliganded and shows an open conformation, suggesting that the enzyme undergoes a major conformational change upon binding of both substrates. The residues involved in binding of the inhibitor and the residues involved in catalysis have been identified on the basis of the three-dimensional structure. Site-directed mutants have been used to further characterize the role of these residues in binding and catalysis. A chemical mechanism for the enzyme, based on these and previously reported data, is proposed.

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

“…Subsequently 2,3-dihydrodipicolinate (DHDP) may be formed spontaneously under physiological conditions (Blickling et al 1997b). It is generally assumed that DHDP is substrate for dihydrodipicolinate reductase (Scapin et al 1997;Wang et al 1997) (Fig. 1).…”

Enzymology of Bacterial Lysine Biosynthesis

“…Kinetic analysis of DHDPR from M. tuberculosis also shows that the enzyme exhibits only a moderate preference for NADH. The crystal structures of M. tuberculosis DHDPR in two ternary complexes (DHDPR-2,6-PDC-NADH and DHDPR-2,6-PDC-NADPH) demonstrate that the number of hydrogen bonds between DHDPR and the nucleotides NADH and NADPH are very similar (Cirilli et al, 2003;Reddy et al, 1996;Scapin et al, 1997).…”

“…The three-dimensional structure of DHDPR has been elucidated by X-ray crystallography from five diverse bacterial species, namely, Bartonella henselae, (PDB: 3IJP), E. coli (Scapin et al, 1995(Scapin et al, , 1997, M. tuberculosis (Cirilli et al, 2003), S. aureus (Girish et al, 2011), and T. maritima . DHDPR from E. coli (Fig.…”

“…DHDPR from E. coli (Fig. 8) was the first DHDPR enzyme to be extensively studied in terms of structure and function (Farkas & Gilvarg, 1965;Reddy et al, 1995;Scapin et al, 1995Scapin et al, , 1997.…”

“…This sequence is thought to contain residues involved in binding of substrate and/or catalysis. Molecular modelling studies, using the apo form (enzyme in the absence of substrate) of E. coli DHDPR as a structural template, suggest a cluster of five basic residues are the key catalytic site residues (Scapin et al, 1997), namely His159, His160, Arg161, His162 and Lys163 (all contained within the consensus sequence). These residues are located in the loop connecting -strand B7 to -helix A5.…”