New insights into the binding mode of coenzymes: structure of Thermus thermophilus Δ¹-pyrroline-5-carboxylate dehydrogenase complexed with NADP⁺

Δ¹-Pyrroline-5-carboxylate dehydrogenase (P5CDh) is known to preferentially use NAD⁺ as a coenzyme. The k_cat value of Thermus thermophilus P5CDh (TtP5CDh) is four times lower for NADP⁺ than for NAD⁺. The crystal structure of NADP⁺-bound TtP5CDh was solved in order to study the structure–activity relationships for the coenzymes. The binding mode of NADP⁺ is essentially identical to that in the previously solved NAD⁺-bound form, except for the regions around the additional 2′-phosphate group of NADP⁺. The coenzyme-binding site can only accommodate this group by the rotation of a glutamate residue and subtle shifts in the main chain. The 2′-phosphate of NADP⁺ increases the number of hydrogen bonds between TtP5CDh and NADP⁺ compared with that between TtP5CDh and NAD⁺. Furthermore, the phosphate of the bound NADP⁺ would restrict the `bending' of the coenzyme because of steric hindrance. Such bending is important for dissociation of the coenzymes. These results provide a plausible explanation of the lower turnover rate of NADP⁺ compared with NAD⁺.