Crystallographic and biophysical analyses of Pseudomonas aeruginosa ketopantoate reductase: Implications of ligand induced conformational changes in cofactor recognition

Abstract

Ketopantoate reductases (KPRs) catalyse NADPH-dependent reduction of ketopantoate to pantoate, the rate-limiting step of pantothenate biosynthetic pathway. In our recent study, we showed KPRs are under dynamic evolutionary selection and highlighted the possible role of ordered substrate binding kinetics for cofactor selection. To further delineate this at molecular level, here, we perform X-ray crystallographic and biophysical analyses of KPR in presence of non-canonical cofactor NAD+. In our structure, NAD+ was found to be highly dynamic in catalytic pocket of KPR, which could attain stable conformation only in presence of ketopantoate. Further, isothermal calorimetric (ITC) titrations showed that affinity of KPR for ketopantoate is higher in presence of NADP+ than in presence of NAD+ and lowest in absence of redox cofactors. In sum, our results clearly depict two modes of redox cofactor selections in KPRs, firstly by specific salt bridge interactions with unique phosphate moiety of NADP+ and secondly via ordered sequential heterotrophic cooperative binding of substrate ketopantoate.