Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP +) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

• Published in: Journal of molecular structure Vol. 602; pp. 257 - 267
• Main Authors: Polshakov, Vladimir I, Biekofsky, Rodolfo R, Birdsall, Berry, Feeney, James
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 09-01-2002
• Subjects: Chemical shift; Density functional theory; Hydrogen bond; Lactobacillus casei dihydrofolate reductase; Nicotinamide adenine dinucleotide phosphate; Nuclear magnetic resonance; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NADP +, oxidised nicotinamide adenine dinucleotide phosphate; Lactobacillus casei dihydrofolate reductase; DFT, Density Functional Theory; DHFR, dihydrofolate reductase; Chemical shift; Nicotinamide adenine dinucleotide phosphate; NMR, nuclear magnetic resonance; LCS, Ligand-induced chemical shift; Density functional theory; GIAO, Gauge-Independent Atomic Orbital; HSQC, heteronuclear single-quantum correlation spectroscopy; Nuclear magnetic resonance; B3LYP, Becke's Three Parameter Hybrid Functional using the Lee, Yang and Parr Correlation Functional; Hydrogen bond
• ISSN: 0022-2860; 1872-8014
• DOI: 10.1016/S0022-2860(01)00687-1

Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP
+. The origins of the difference in binding affinity to DHFR between NADPH and NADP
+ are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in
1H and
15N chemical shift between NADPH and NADP
+ in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP
+ compared with NADPH results partly from NADP
+ having to change its nicotinamide amide group from a
cis- to a
trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP
+ being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.