Structural insights into unique substrate selectivity of Thermoplasma acidophilum D-aldohexose dehydrogenase.
The D-aldohexose dehydrogenase from the thermoacidophilic archaea Thermoplasma acidophilum (AldT) belongs to the short-chain dehydrogenase/reductase (SDR) superfamily and catalyzes the oxidation of several monosaccharides with a preference for NAD(+) rather than NADP(+) as a cofactor. It has been found that AldT is a unique enzyme that exhibits the highest dehydrogenase activity against D-mannose. Here, we describe the crystal structures of AldT in ligand-free form, in complex with NADH, and in complex with the substrate D-mannose, at 2.1 A, 1.65 A, and 1.6 A resolution, respectively. The AldT subunit forms a typical SDR fold with an unexpectedly long C-terminal tail and assembles into an intertwined tetramer. The D-mannose complex structure reveals that Glu84 interacts with the axial C2 hydroxyl group of the bound D-mannose. Structural comparison with Bacillus megaterium glucose dehydrogenase (BmGlcDH) suggests that the conformation of the glutamate side-chain is crucial for discrimination between D-mannose and its C2 epimer D-glucose, and the conformation of the glutamate side-chain depends on the spatial arrangement of nearby hydrophobic residues that do not directly interact with the substrate. Elucidation of the D-mannose recognition mechanism of AldT further provides structural insights into the unique substrate selectivity of AldT. Finally, we show that the extended C-terminal tail completely shuts the substrate-binding pocket of the neighboring subunit both in the presence and absence of substrate. The elaborate inter-subunit interactions between the C-terminal tail and the entrance of the substrate-binding pocket imply that the tail may play a pivotal role in the enzyme activity.
Yasutake
Yoshiaki
Y
Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, Toyohira-ku, Sapporo 062-8517, Japan.
Nishiya
Yoshiaki
Y
Tamura
Noriko
N
Tamura
Tomohiro
T
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology; D001665:Binding Sites; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013822:Thermoplasma / Q000201:enzymology*
D003067:Coenzymes; D021122:Protein Subunits; D002237:Carbohydrate Dehydrogenases; C501231:aldohexose dehydrogenase; D005947:Glucose; D008358:Mannose