Investigation of the functional relevance of the catalytically important Glu(28) in family 51 arabinosidases.
The alpha-L-arabinofuranosidase (AbfD3) from Thermobacillus xylanilyticus is a family 51 glycosyl hydrolase. According to classification hierarchy, family 51 belongs to clan GH-A. While the major GH-A motifs, the catalytic acid-base and nucleophile, are conserved in AbfD3, a third catalytically important residue (Glu(28)) does not appear to be analogous to any known GH-A motif. To evaluate the importance of Glu(28), bioinformatics analyses and site-saturation mutagenesis were performed. The results indicate that Glu(28) forms part of a family 51 arabinosidase motif which might be functionally homologous to a conserved N-terminal motif found in exo-acting enzymes from families 1 and 5. Importantly, the data reveal that Glu(28) is a key determinant of substrate recognition in the -1 subsite, where it may also play an important role in water-mediated deglycosylation of the glycosyl-enzyme covalent intermediate.
Institut National de la Recherche Agronomique, UMR FARE, 8, rue Gabriel Voisin, P.O. Box 316, 51688 Cedex 2 Reims, France.
Mounir
M
Ferchichi
Caroline
C
Rémond
Roselyne
R
Simo
Michael J
MJ
O'Donohue
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000201:enzymology; D020134:Catalytic Domain; D019295:Computational Biology / Q000379:methods; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D011994:Recombinant Proteins; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase