Interaction of Pyridine Nucleotide Substrates with Escherichia coli Dihydrodipicolinate Reductase:  Thermodynamic and Structural Analysis of Binary Complexes^,

Abstract

E. coli dihydrodipicolinate reductase exhibits unusual nucleotide specificity, with NADH being kinetically twice as effective as NADPH as a reductant as evidenced by their relative V/K values. To investigate the nature of the interactions which determine this specificity, we performed isothermal titration calorimetry to determine the thermodynamic parameters of binding and determined the three-dimensional structures of the corresponding enzyme-nucleotide complexes. The thermodynamic binding parameters for NADPH and NADH were determined to be Kd = 2.12 microM, delta G degree = -7.81 kcal mol-1, delta H degree = -10.98 kcal mol-1, and delta S degree = -10.5 cal mol-1 deg-1 and Kd = 0.46 microM, delta G degree = -8.74 kcal mol-1, delta H degree = -8.93 kcal mol-1, and delta S degree = 0.65 cal mol-1 deg-1, respectively. The structures of DHPR complexed with these nucleotides have been determined at 2.2 A resolution. The 2'-phosphate of NADPH interacts electrostatically with Arg39, while in the NADH complex this interaction is replaced by hydrogen bonds between the 2' and 3' adenosyl ribose hydroxyls and Glu38. Similar studies were also performed with other pyridine nucleotide substrate analogs to determine the contributions of individual groups on the nucleotide to the binding affinity and enthalpic and entropic components of the free energy of binding, delta G degree. Analogs lacking the 2'-phosphate containing homologs. For all analogs, the total binding free energy can be shown to include compensating enthalpic and entropic contributions to the association constants. The entropy contribution appears to play a more important role in the binding of the nonphosphorylated analogs than in the binding of the phosphorylated analogs.

Characterization of Determinants of Ligand Binding to the Nicotinic Acid Receptor GPR109A (HM74A/PUMA-G)

“…From analyses of crystal structures of nicotinic acid bound at diverse prokaryotic proteins [such as nicotinate mononucleotide dimethylbenzimidazole phosphoribosyltransferase (PDB code 1D0V) (Cheong et al, 1999), dihydropteridine reductase (PDB code 1ICR) (Lovering et al, 2001), nicotinate nucleotide dimethylbenzimidazole phosphoribosyltransferase (PDB code 1JHA) (Cheong et al, 2001), dihydrodipicolinate reductase (PDB code 1DRV) (Reddy et al, 1996), and the plant protein ferric soybean leghemoglobin (PDB code 1FSL) (Ellis et al, 1997)], it is evident that the pyridine ring system of the ligand is always bound near aromatic side chains of the protein. Following the structural homology paradigm, we assumed that the binding pocket of GPR109A is also coated by aromatic side chain(s) as an additional binding partner for nicotinic acid.…”

The Negative Transcriptional Regulator NmrA Discriminates between Oxidized and Reduced Dinucleotides

“…Whereas this discrimination cannot be put down to a particular common set of residues, nevertheless "fingerprint" interactions for NAD ϩ (a side-chain carboxylate interacting with the adenosine ribose hydroxyls) and for NADP ϩ (an arginine facing the adenine plane and interacting with the pyrophosphate) have been identified (32,33). Unusually dihydrodipicolinate reductase shows a relatively low discrimination between NADH and NADPH of 4-fold, and in this case the carboxylate of Glu-38 interacts with the adenosine ribose hydroxyls of NADH but does not make this interaction in the NADPH complex (34). The dihydrodipicolinate reductase NADPH complex has an interaction that is not present in the NADH complex involving Arg-39.…”

Specificity determinants for lipids bound to β-barrel proteins

“…Generally, most enthalpic contributions arise from the balance of noncovalent bonding, such as various polar interactions. The burial of nonpolar residues, solvent reorganization, and significant structural changes have been associated more with systems of entropic character (51)(52)(53).…”