Crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus

The crystal structure of glycosyltrehalose trehalohydrolase from the hypert hermophilic archaeum Sulfolobus solfataricus KM1 has been solved by multipl e isomorphous replacement. The enzyme is an a-amylase (family 13) with uniq ue exo-amylolytic activity for glycosyltrehalosides. It cleaves the alpha-1 ,4 glycosidic bond adjacent to the trehalose moiety to release trehalose an d maltooligo saccharide. Unlike most other family 13 glycosidases, the enzy me does not require Ca2+ for activity, and it contains an N-terminal extens ion of similar to 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 li nked 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 connecte d to the catalytic domain by an extended linker. The functionally essential Ca2+ binding site found in the B domain of alpha-amylases and many other f amily 13 glycosidases was found to be replaced by hydrophobic packing inter actions. The enzyme also contains a very unusual excursion in the (beta/alp ha)(8) barrel structure of the catalytic domain. This excursion originates from the bottom of the (beta/alpha)(8) barrel between helix 6 and strand 7, but folds upward in a distorted a-hairpin structure to form a part of the substrate binding cleft wall that is possibly critical for the enzyme's uni que substrate selectivity. Participation of an a-p loop in the formation of the substrate binding cleft is a novel feature that is not observed in oth er known (beta/alpha)(8) enzymes. (C) 2000 Academic Press.