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

Abstract

Ferroplasma acidiphilum strain Y (DSM 12658), a ferrous iron-oxidizing, acidophilic and mesophilic archaeon, was found to produce a membrane-bound α-glucosidase (αGluFa) 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 α-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 (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-α-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 αGluFa is a new member of the glycosyl hydrolase family that uses a novel mechanism for sugar glycosylation and/or transglycosylation.