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 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.
Ferrer
Manuel
M
Department of Microbiology, German Research Centre for Biotechnology (GFB), Mascheroder Weg 1, 38124 Braunschweig, Germany. mferrer@icp.csic.es
Golyshina
Olga V
OV
Plou
Francisco J
FJ
Timmis
Kenneth N
KN
Golyshin
Peter N
PN
D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005960:Glucosides / Q000378:metabolism; D065089:Glycoside Hydrolase Inhibitors; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism*
D005960:Glucosides; D065089:Glycoside Hydrolase Inhibitors; D014312:Trisaccharides; C019502:4-nitrophenyl alpha-glucoside; D013395:Sucrose; C008317:maltotriose; D008320:Maltose; D007501:Iron; D000520:alpha-Glucosidases