Recombinant NAD+-dependent ribitol dehydrogenase derived from Klebsiella oxytoca (KoRdh) exhibits activity toward both ribitol and allitol. KoRdh catalyzes the NAD+-dependent oxidation of allitol to d-allulose and the NADH-dependent reduction of d-allulose to allitol. Notably, the flexible loop of KoRdh undergoes conformational changes upon NAD+ and substrate binding. To elucidate the flexible loop's role in substrate recognition, we determined the X-ray structures of KoRdh alone and in complexes with NAD+, d-allulose, or d-allose. Although d-allose is an aldose and not a substrate of KoRdh, it binds to KoRdh in the pyranose form, revealing the location of the substrate-binding site. Based on these structures, we propose a substrate recognition mechanism for KoRdh. Impact statement This research reveals an insight into a substrate recognition mechanism in the flexible region of ribitol dehydrogenase. Because ribitol dehydrogenase is a member of the short-chain reductases/oxidases (SDR) family, the current study will provide further insight into related enzymes that harbor the flexible region.
Yoshida et al. (Tue,) studied this question.