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ABSTRACT: Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites -1 and +1 glucose.
Protein Science 02/2012; 21(4):539-52. · 2.80 Impact Factor
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ABSTRACT: A new process for trehalose production from starch was developed using a novel glycosyltransferase and a novel α-amylase from Sulfolobus solfataricus KM1. The yield of trehalose from starch was 81.5% using the two enzymes and a thermostable debranching enzyme. Trehalose production was carried out at high temperature over 60°C and at a high concentration of starch with no risk of contamination by microorganisms or retrogradation of starch.
Journal of Fermentation and Bioengineering.
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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 Ca2+ 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 Ca2+ 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.
Journal of Molecular Biology.
