Mannitol‐1‐phosphate dehydrogenases/phosphatases: a family of novel bifunctional enzymes for bacterial adaptation to osmotic stress

Summary The nutritionally versatile soil bacterium A cinetobacter baylyi   ADP 1 copes with salt stress by the accumulation of compatible solutes, a strategy that is widespread in nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to osmotic stress. In a previous study, we identified MtlD , a mannitol‐1‐phosphate dehydrogenase, which is essential for mannitol biosynthesis and which catalyses the first step in mannitol biosynthesis, the reduction of fructose‐6‐phosphate ( F ‐6‐ P ) to the intermediate mannitol‐1‐phosphate ( Mtl ‐1‐ P ). Until now, the identity of the second enzyme, the phosphatase that catalyses the dephosphorylation of Mtl ‐1‐ P to mannitol, was elusive. Here we show that MtlD has a unique sequence among known mannitol‐1‐phosphate dehydrogenases with a haloacid dehalogenase ( HAD )‐like phosphatase domain at the N ‐terminus. This domain is indeed shown to have a phosphatase activity. Phosphatase activity is strictly Mg 2+ dependent. Nuclear magnetic resonance analysis revealed that purified MtlD catalyses not only reduction of F ‐6‐ P but also dephosphorylation of Mtl ‐1‐ P . MtlD of A . baylyi is the first bifunctional enzyme of mannitol biosynthesis that combines Mtl ‐1‐ P dehydrogenase and phosphatase activities in a single polypeptide chain. Bioinformatic analysis revealed that the bifunctional enzyme is widespread among A cinetobacter strains but only rarely present in other phylogenetic tribes.