Reversal of the extreme coenzyme selectivity of Clostridium symbiosum glutamate dehydrogenase

Active‐site mutants of glutamate dehydrogenase from Clostridium symbiosum have been designed and constructed and the effects on coenzyme preference evaluated by detailed kinetic measurements. The triple mutant F238S/P262S/D263K shows complete reversal in coenzyme selectivity from NAD(H) to NADP(H) with retention of high levels of catalytic activity for the new coenzyme. For oxidized coenzymes, kcat/Km ratios of the wild‐type and triple mutant enzyme indicate a shift in preference of approximately 1.6 × 107‐fold, from ∼ 80 000‐fold in favour of NAD+ to ∼ 200‐fold in favour of NADP+. For reduced coenzymes the corresponding figure is 1.7 × 104‐fold, from ∼ 1000‐fold in favour of NADH to ∼ 17‐fold in favour of NADPH. A fourth mutation (N290G), previously identified as having a potential bearing on coenzyme specificity, did not engender any further shift in preference when incorporated into the triple mutant, despite having a significant effect when expressed as a single mutant. In clostridial glutamate dehydrogenase the 2′‐OH of the adenine ribose of NAD+ points into a pocket flanked by F238 and P262 (magenta). The additional phosphate in NADP+ (turquoise) finds an unfavourably hydrophobic pocket, insufficient space, possible repulsion by D263 and hindrance by N290 (both magenta). Triple mutant F238S/P262S/D263K relieves these constraints and reverses coenzyme specificity