A novel alpha-glucosidase from the acidophilic archaeon Ferroplasma acidiphilum strain Y with high transglycosylation activity and an unusual catalytic nucleophile

Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound alpha-glucosidase (alphaGluFa) showing no significant similarity to any of the known glycoside hydrolases classified in different families and having an...

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Bibliographic Details
Published in:Biochemical journal 2005-10, Vol.391 (Pt 2), p.269-276
Main Authors: Ferrer, Manuel, Golyshina, Olga V, Plou, Francisco J, Timmis, Kenneth N, Golyshin, Peter N
Format: Article
Language:English
Subjects:
alpha-Glucosidases - genetics
alpha-Glucosidases - metabolism
Archaea - enzymology
Archaea - genetics
Binding Sites
Catalysis
Cloning, Molecular
Enzyme Stability
Ferroplasma
Glucosides - metabolism
Glycoside Hydrolase Inhibitors
Glycosylation
Hydrogen-Ion Concentration
Iron - metabolism
Kinetics
Maltose - metabolism
Protein Binding
Substrate Specificity
Sucrose - metabolism
Temperature
Trisaccharides - metabolism
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Summary:Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound alpha-glucosidase (alphaGluFa) showing no significant similarity to any of the known glycoside hydrolases classified in different families and having an unusual catalytic site consisting of a threonine and a histidine residue. The highest alpha-glucosidase activity was found at low pH, 2.4-3.5, and the substrate preference order was: sucrose>maltose>maltotriose >>maltotetraose>>malto-oligosaccharides from maltopentaose to maltoheptaose>>>soluble starch (kcat/K(m) was 293.0, 197.0, 18.8, 0.3 and 0.02 s(-1) x mM(-1) respectively). The enzyme was able to transfer glucosyl groups from maltose as donor, to produce exclusively maltotriose (up to 300 g/l). Chemical modification and electrospray ionization MS analysis of 5-fluoro-alpha-D-glucopyranosyl-enzyme derivatives, coupled with site-directed mutagenesis, strongly suggested that the putative catalytic nucleophile in this enzyme is Thr212. Iron was found to be essential for enzyme activity and integrity, and His390 was shown to be essential for iron binding. These results suggest that the metalloenzyme alphaGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.
ISSN:0264-6021
1470-8728
DOI:10.1042/BJ20050346