A novel α-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 archacon, was found to produce a membrane-bound a-glucosidase (alpha GluFa) 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 a-glucosidase activity was found at low pH, 2.4-3.5, and the substrate preference order was: sucrose > maltose > maltotriose >> maltotetraose >> mal to-oligosaccharides from maltopentaose to maltoheptaose >>> soluble starch (k(cat)/K-m was 293.0, 197.0, 18.8, 0.3 and 0.02 s(-1.)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 Thr(212). Iron was found to be essential for enzyme activity and integrity, and His(390). was shown to be essential for iron binding. These results suggest that the metalloenzyme aGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.