Substrate (aglycone) specificity of human cytosolic beta-glucosidase.
Human cytosolic beta-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays beta-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (beta/alpha)(8)-barrel fold characteristic of family 1 beta-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val(168)-->Tyr, Phe(225)-->Ser, Tyr(308)-->Ala and Tyr(308)-->Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val(168)-->Tyr mutation did not affect K (m) on p -nitrophenyl ( p NP)-glycosides, but increased K (m) 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe(225)-->Ser and Tyr(308)-->Ala mutations, and, to a lesser degree, the Tyr(308)-->Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.
Berrin
Jean-Guy
JG
Institute of Food Research, Colney Lane, Norwich NR4 7UA, UK.
Czjzek
Mirjam
M
Kroon
Paul A
PA
McLauchlan
W Russell
WR
Puigserver
Antoine
A
Williamson
Gary
G
Juge
Nathalie
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D001617:beta-Glucosidase