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.

Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat.

We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem. J. 338, 441-446]. In the present study, we show that XIP-I inhibits family-10 and -11 fungal xylanases. The K(i) values for fungal xylanases ranged from 3.4 to 610 nM, but bacterial family-10 and -11 xylanases were not inhibited. Unlike many glycosidase inhibitors, XIP-I was not a slow-binding inhibitor of the Aspergillus niger xylanase. Isothermal titration calorimetry of the XIP-I-A. niger xylanase complex showed the formation of a stoichiometric (1:1) complex with a heat capacity change of -1.38 kJ x mol(-1) x K(-1), leading to a predicted buried surface area of approx. 2200+/-500 A(2) at the complex interface. For this complex with A. niger xylanase (K(i)=320 nM at pH 5.5), titration curves indicated that an observable interaction occurred at pH 4-7, and this was consistent with the pH profile of inhibition of activity. In contrast, the stronger complex between A. nidulans xylanase and XIP-I (K(i)=9 nM) led to an observable interaction across the entire pH range tested (3-9). Using surface plasmon resonance, we show that the differences in the binding affinity of XIP-I for A. niger and A. nidulans xylanase are due to a 200-fold lower dissociation rate k(off) for the latter, with only a small difference in association rate k(on).

Functional expression of human liver cytosolic beta-glucosidase in Pichia pastoris. Insights into its role in the metabolism of dietary glucosides.

Human tissues such as liver, small intestine, spleen and kidney contain a cytosolic beta-glucosidase (CBG) that hydrolyses various beta-d-glycosides, but whose physiological function is not known. Here, we describe the first heterologous expression of human CBG, a system that facilitated a detailed assessment of the enzyme specificity towards dietary glycosides. A full-length CBG cDNA (cbg-1) was cloned from a human liver cDNA library and expressed in the methylotrophic yeast Pichia pastoris at a secretion yield of approximately 10 mg x L-1. The recombinant CBG (reCBG) was purified from the supernatant using a single chromatography step and was shown to be similar to the native enzyme isolated from human liver in terms of physical properties and specific activity towards 4-nitrophenyl-beta-D-glucoside. Furthermore, the reCBG displayed a broad specificity with respect to the glycone moiety of various aryl-glycosides (beta-D-fucosides, alpha-L-arabinosides, beta-D-glucosides, beta-D-galactosides, beta-L-xylosides, beta-D-arabinosides), similar to the native enzyme. For the first time, we show that the human enzyme has significant activity towards many common dietary xenobiotics including glycosides of phytoestrogens, flavonoids, simple phenolics and cyanogens with higher apparent affinities (K(m)) and specificities (k(cat)/K(m)) for dietary xenobiotics than for other aryl-glycosides. These data indicate that human CBG hydrolyses a broad range of dietary glucosides and may play a critical role in xenobiotic metabolism.