Molecular insights into the regulation of coenzyme selection in NADP-specific glutamate dehydrogenase from Saccharomyces cerevisiae

Abstract

Coenzyme specificity is one of the crucial aspects of the functional regulations in enzymes. Understanding the molecular basis of coenzyme specificity in glutamate dehydrogenase (GDH) is highly essential as this is the only enzyme that fulfils various physiological demands in terms of energy production and/or nitrogen assimilation via its strict functional regulation by connecting carbon and nitrogen metabolic cycles in every living organism. We have studied S. cerevisiae GDH3 (ScGDH3), as a model system for NADP-specific fungal GDHs. K272R and S250E mutants of ScGDH3 exhibited remarkable alteration in coenzyme specificity, with 15-55-fold and 3-14-fold more preference towards non-specific coenzymes [NAD(H)], respectively. S250E exhibited higher NADH affinity (~ 1.3-fold) than that of towards NADPH. K272R showed ~ 10-fold increased coenzyme affinity (1/KM) and binding affinity (1/kd) towards NADH, compared to the wild type enzyme (ScGDH3-WT). We solved the crystal structure of ScGDH3-K272R complexed with NADP+ at 2.50 [A] resolution with a unique oligomeric arrangement having two distinct subunit conformations (''open'' and ''super-closed'') and it is the first structure of a GDH from S. cerevisiae. Molecular dynamics (MD) simulations provide insights into NADH binding and its increased affinity to ScGDH3-K272R. Further structure-based sequence analyses unravelled that a lysine and an arginine are naturally preferred residues for providing NADP(H) and NAD(H) specificity, respectively for various amino acid dehydrogenases. Taken together biochemical and structural data, our study successfully illustrates the evolutionary conserved mechanism of coenzyme related regulation in fungal and other GDHs; as well as amino acid dehydrogenases.