Structural insights into broad substrate specificity and catalytic activity of Thermoplasma acidophilum aldohexose dehydrogenase

Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST)* Tsuruga Institute of Biotechnology, Toyobo Co. Ltd, Japan**
â—‹Yoshiaki Yasutake* Yoshiaki Nishiya** Noriko Tamura* Tomohiro Tamura*

The aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is an enzyme that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily, and catalyzes the oxidation of C1 hydroxyl of various monosaccharides such as D-mannose and D-xylose, with a preference of NAD+ rather than NADP+ as a cofactor. Most of the functionally related enzymes in the SDR superfamily are found in Bacillus species and display the highest activity for D-glucose. In contrast, AldT exhibits very low activity for D-glucose but the highest activity against D-mannose. To date, AldT is the only enzyme known to display an efficient NAD+-dependent dehydration activity against D-mannose. Although several crystal structures of the SDR family enzymes were determined, no structural information regarding the monosaccharide-recognition mechanism is available. It is of interest to investigate how these enzymes discriminate between various monosaccharides, particularly D-glucose and its C2 epimer D-mannose. AldT crystallized in space group P3221 with unit-cell dimensions of a = b = 82.0 and c =138.7 Å, and the structures of AldT in ligand-free form, cofactor complex, and substrate complex were determined by the molecular replacement method using Bacillus megaterium glucose dehydrogenase as a search model. The AldT monomer forms a single domain structure with an unexpected long C-terminal tail, and assembles into intertwined tetramer having a 222 point-group symmetry. The details of the structure, structural mechanism of cofactor and monosaccharide recognition, and the significance of the C-terminal tail of the enzyme will be presented.