Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana

Abstract

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD +, Mn 2+, and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0 Å in the presence of Mn 2+ but in the absence of NAD +. The structure showed the dimeric assembly and the Mn 2+ coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD +, which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.