Abstract
The crystal structure of Escherichia coli ketopantoate reductase in complex with 2'-monophosphoadenosine 5'-diphosphoribose, a fragment of NADP+ that lacks the nicotinamide ring, is reported. The ligand is bound at the enzyme active site in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket. Isothermal titration calorimetry with R31A and N98A mutants of the enzyme is used to show that the unusual ;reversed binding mode' observed in the crystal is triggered by changes in the protonation of binding groups at low pH. This research has important implications for fragment-based approaches to drug design, namely that the crystallization conditions and the chemical modification of ligands can have unexpected effects on the binding modes.
Highlights
Ketopantoate reductase (KPR; EC 1.1.1.169), an enzyme involved in the biosynthesis of pantothenate, catalyses the reduction of ketopantoate to form pantoate using NADPH as a cofactor (Ciulli & Abell, 2005)
We report the three-dimensional structure of KPR in complex with 20-monophosphoadenosine 50-diphosphoribose (20P-ADP-ribose), a fragment of NADPH that lacks the nicotinamide ring, solved to 1.95 Aresolution
The protein concentration was determined from the A280, using an absorption coefficient for His6-KPR of 62 650 MÀ1 cmÀ1 determined from aminoacid analysis [Protein and Nucleic Acid Chemistry Facility (PNAC), Cambridge]
Summary
Ketopantoate reductase (KPR; EC 1.1.1.169), an enzyme involved in the biosynthesis of pantothenate (vitamin B5), catalyses the reduction of ketopantoate to form pantoate using NADPH as a cofactor (Ciulli & Abell, 2005). We have solved the crystal structure of KPR in complex with NADP+ (Lobley et al, 2005) This structure showed the cofactor bound in an extended conformation in the cleft between the two domains and identified the interactions formed with key residues at the enzyme active site (Lobley et al, 2005). The structure surprisingly reveals the ligand to be bound in the opposite orientation to that observed for NADP+, with the adenine ring occupying the lipophilic nicotinamide pocket Detailed analysis of this unexpected crystallographic result using isothermal titration calorimetry (ITC) and site-directed mutagenesis revealed that this unusual ‘reversed binding mode’ is triggered by changes in the protonation of binding groups at low pH
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