Substrate (aglycone) specificity of human cytosolic β-glucosidase

被引:63
作者
Berrin, JG
Czjzek, M
Kroon, PA
McLauchlan, WR
Puigserver, A
Williamson, G
Juge, N
机构
[1] Food Res Inst, Norwich NR4 7UA, Norfolk, England
[2] Fac Sci & Tech St Jerome, Inst Mediterraneen Rech Nutr, INRA, UMR 1111, F-13397 Marseille 20, France
[3] CNRS, AFMB, UMR 6098, F-13402 Marseille 20, France
关键词
binding subsite; flavonoid glycosides; glycosyl hydrolase family 1; site-directed mutagenesis; three-dimensional model;
D O I
10.1042/BJ20021876
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (pNP)-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.
引用
收藏
页码:41 / 48
页数:8
相关论文
共 39 条
[1]   Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: Resilience as a key factor in thermostability [J].
Aguilar, CF ;
Sanderson, I ;
Moracci, M ;
Ciaramella, M ;
Nucci, R ;
Rossi, M ;
Pearl, LH .
JOURNAL OF MOLECULAR BIOLOGY, 1997, 271 (05) :789-802
[2]  
[Anonymous], SILICON GRAPHICS GEO
[3]   Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase [J].
Arribas, JCD ;
Herrero, AG ;
Martín-Lomas, M ;
Cañada, FJ ;
He, SM ;
Withers, SG .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 2000, 267 (24) :6996-7005
[4]   THE CRYSTAL-STRUCTURE OF A CYANOGENIC BETA-GLUCOSIDASE FROM WHITE CLOVER, A FAMILY-1 GLYCOSYL HYDROLASE [J].
BARRETT, T ;
SURESH, CG ;
TOLLEY, SP ;
DODSON, EJ ;
HUGHES, MA .
STRUCTURE, 1995, 3 (09) :951-960
[5]   PROTEIN DATA BANK - COMPUTER-BASED ARCHIVAL FILE FOR MACROMOLECULAR STRUCTURES [J].
BERNSTEIN, FC ;
KOETZLE, TF ;
WILLIAMS, GJB ;
MEYER, EF ;
BRICE, MD ;
RODGERS, JR ;
KENNARD, O ;
SHIMANOUCHI, T ;
TASUMI, M .
JOURNAL OF MOLECULAR BIOLOGY, 1977, 112 (03) :535-542
[6]   Functional expression of human liver cytosolic β-glucosidase in Pichia pastoris -: Insights into its role in the metabolism of dietary glucosides [J].
Berrin, JG ;
McLauchlan, WR ;
Needs, P ;
Williamson, G ;
Puigserver, A ;
Kroon, PA ;
Juge, N .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 2002, 269 (01) :249-258
[7]   The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase [J].
Burmeister, WP ;
Cottaz, S ;
Driguez, H ;
Iori, R ;
Palmieri, S ;
Henrissat, B .
STRUCTURE, 1997, 5 (05) :663-675
[8]   Crystal structure of the β-glycosidase from the hyperthermophile Thermosphaera aggregans:: insights into its activity and thermostability [J].
Chi, YI ;
Martinez-Cruz, LA ;
Jancarik, J ;
Swanson, RV ;
Robertson, DE ;
Kim, SH .
FEBS LETTERS, 1999, 445 (2-3) :375-383
[9]   PICHIA-PASTORIS AS A HOST SYSTEM FOR TRANSFORMATIONS [J].
CREGG, JM ;
BARRINGER, KJ ;
HESSLER, AY ;
MADDEN, KR .
MOLECULAR AND CELLULAR BIOLOGY, 1985, 5 (12) :3376-3385
[10]   Crystal structure of a monocotyledon (maize ZMGlu1) β-glucosidase and a model of its complex with p-nitrophenyl β-D-thioglucoside [J].
Czjzek, M ;
Cicek, M ;
Zamboni, V ;
Burmeister, WP ;
Bevan, DR ;
Henrissat, B ;
Esen, A .
BIOCHEMICAL JOURNAL, 2001, 354 :37-46