Crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus.

Abstract The crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus KM1 has been solved by multiple isomorphous replacement. The enzyme is an α-amylase (family 13) with unique exo-amylolytic activity for glycosyltrehalosides. It cleaves the α-1,4 glycosidic bond adjacent to the trehalose moiety to release trehalose and maltooligo saccharide. Unlike most other family 13 glycosidases, the enzyme does not require Ca 2+ for activity, and it contains an N-terminal extension of ∼100 amino acid residues that is homologous to N-terminal domains found in many glycosidases that recognize branched oligosaccharides. Crystallography revealed the enzyme to exist as a homodimer covalently linked by an intermolecular disulfide bond at residue C298. The existence of the intermolecular disulfide bond was confirmed by biochemical analysis and mutagenesis. The N-terminal extension forms an independent domain connected to the catalytic domain by an extended linker. The functionally essential Ca 2+ binding site found in the B domain of α-amylases and many other family 13 glycosidases was found to be replaced by hydrophobic packing interactions. The enzyme also contains a very unusual excursion in the (β/α) 8 barrel structure of the catalytic domain. This excursion originates from the bottom of the (β/α) 8 barrel between helix 6 and strand 7, but folds upward in a distorted α-hairpin structure to form a part of the substrate binding cleft wall that is possibly critical for the enzyme’s unique substrate selectivity. Participation of an α-β loop in the formation of the substrate binding cleft is a novel feature that is not observed in other known (β/α) 8 enzymes.