Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase (original) (raw)
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Amino acid residues involved in substrate binding and catalysis in an insect digestive β-glycosidase
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 2001
A L-glycosidase (M r 50 000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl L-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK a1 = 4.9 and pK a2 = 7.5). Effect of pH on carbodiimide inactivation indicates that the pK a 7.5 group is a carboxyl. k cat and K m values were obtained for different p-nitrophenyl L-glycosides and K i values were determined for a range of alkyl L-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature L-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E 187 (proton donor) and E 399 (nucleophile) ; (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite. ß 0167-4838 / 01 / $^see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 -4 8 3 8 ( 0 0 ) 0 0 2 6 0 -0 Abbreviations: M r , relative molecular
Insect Biochemistry and Molecular Biology, 2000
Two β-glycosidases (BG) (Mr 47,000 and Mr 50,000) were purified from Spodoptera frugiperda (Lepidoptera: Noctuidae) midguts. These two polypeptides associate or dissociate depending on the medium ionic strength. The Mr 47,000 BG probably has two active sites. One of the putative active sites (cellobiase site) hydrolyses p-nitrophenyl β-d-glucoside (NPβGlu) (79% of the total activity in saturated enzyme), cellobiose, amygdalin and probably also cellotriose, cellotetraose and cellopentaose. The cellobiase site has four subsites for glucose residue binding, as can be deduced from cellodextrin cleavage data. The enzymatic activity in this site is abolished after carbodiimide modification at pH 6.0. Since the inactivation is reduced in the presence of cellobiose, the results suggest the presence of a carboxylate as a catalytic group. The other active site of Mr 47,000 BG (galactosidase site) hydrolyses p-nitrophenyl β-d-galactoside (NPβGal) better than NPβGlu, cleaves glucosylceramide and lactose and is unable to act on cellobiose, cellodextrins and amygdalin. This active site is not modified by carbodiimide at pH 6.0.
Insect Biochemistry and Molecular Biology, 2001
Two β-glycosidases (M r 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (βGly1 and βGly2) have identical kinetic properties, but differ in hydrophobicity. The two glycosidases were cloned and their sequences differ by only four amino acids. The T. molitor glycosidases are family 1 glycoside hydrolases and have the E 379 (nucleophile) and E 169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant β-glycosidases. The two enzymes may hydrolyze several substrates, such as disaccharides, arylglucosides, natural occurring plant glucosides, alkylglucosides, oligocellodextrins and the polymer laminarin. The enzymes have only one catalytic site, as inferred from experiments of competition between substrates and sequence alignments. The observed inhibition by high concentrations of the plant glucoside amygdalin, used as substrate, is an artifact generated by transglucosylation. The active site of each purified β-glycosidase has four subsites, of which subsites +1 and +2 bind glucose with more affinity. Subsite +2 has more affinity for hydrophobic groups, binding with increasing affinities: glucose, mandelonitrile and nitrophenyl moieties. Subsite +3 has more affinity for glucose than butylene moieties. The intrinsic catalytic constant calculated for hydrolysis of the glucose β-1,4-glucosidic bond is 21.2 s Ϫ1 M Ϫ1 . The putative physiological role of these enzymes is the digestion of di-and oligosaccharides derived from hemicelluloses.
FEBS Journal, 2008
The b-glycosidases from family 1 of the glycoside hydrolases are widely distributed among living organisms, being found in bacteria, archea and eukaria. These enzymes are involved in a high diversity of physiological roles . b-glycosidases catalyze the hydrolytic removal of the monosaccharide from the non-reducing end of b-glycosides [2,3]. Their active site may be divided into subsites, which are of sufficient size to bind a monosaccharide unit. The monosaccharide forming the non-reducing end of the substrate, called glycone, is bound at subsite )1, whereas the remaining part of the substrate, called aglycone, interacts with the aglycone-binding site, which may be composed of several subsites identified by positive numerals. The substrate is cleaved between subsites )1 and +1 .
Insect Biochemistry and Molecular Biology, 2003
Three β-glycosidases, named βGly1, βGly2 and βGly3, were isolated from midgut tissues of the sugar cane borer, Diatraea saccharalis Fabricius (Lepidoptera: Pyralidae). The three enzymes have similar Mr (58,000; 61,000; 61,000), pI (7.5, 7.4, and 7.4) and optimum pH (6.7, 6.3, and 7.2) and were resolved by hydrophobic chromatography. The β-glycosidases prefer β-glucosides to β-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose β-1,3 linkages better than β-1, 4-or β-1,6 linkages. βGly1 and 2 were completely purified, whereas βGly3 was isolated with a contaminant peptide that has no activity upon β-glycosides.
Insect Biochemistry and Molecular Biology, 1995
A combination of gel filtration, ion-exchange chromatography, polyacrylamide gel electrophoresis, and heat inactivation data revealed the existence of three /I-glucosidases with M, 82,000 in Abracris flavolineata midgut contents: 1, a major heat-stable activity against celiobiose (ceilobiasearyl/]-glucosidase); 2, a minor heat-unstable activity against p-nitrophenyi/l-D-glucoside (NP~Glu) (aryl/I-glucosidase); 3, an activity against octyl-~-glucoside (alkylel-glucosidase). The cellobiase-aryl-/Iglucosidase has a pH optimum of 5.5 and is more active on cellobiose and laminaribiose than on synthetic or natural aryl~-glucosides. Experiments involving competition between substrates and the use of inhibitors suggested that ceHobiase-aryl/I-glucosidase hydrolyzes cellobiose and aryl/I-glucosides at different active sites. Alkyl ~-glucosidase (pH optimum 4.8) has a sigmoidal activity-octyl /]-glucoside-concentration profile, which changes to a hyperbolic profile in the presence of excess Triton X-100. NP/I Glu, which is hydrolyzed at the same site as octyi/I-glucoside, has a hyperbolic activity-NP~Glu-concentration profile that increases in the presence of Triton X-100. It seems that amphipathic molecules activate the alkyl~-glucosidase, which is inactive on methylp-D-ghicoside and is most active on CT--Ct0 alkyl-/lglucosides. The aryl/I-glucosidase activity of the ceHobiase-arylll-glucosidase and the alkyl/I-glucosidase are probably responsible for in vivo digestion of ~ 1,3-glucans and glucosylceramides, respectively. Activation by detergent-like molecules is supposed to maintain high alkyl/I-glucosidase activity only during plant cell membrane digestion. This avoids extensive hydrolysis of toxic plant/]-glucosides which may be ingested by the insects.
Using the Amino Acid Network to Modulate the Hydrolytic Activity of β-Glycosidases
PLOS ONE
The active site residues in GH1 β-glycosidases are compartmentalized into 3 functional regions, involved in catalysis or binding of glycone and aglycone motifs from substrate. However, it still remains unclear how residues outside the active site modulate the enzymatic activity. To tackle this question, we solved the crystal structure of the GH1 β-glycosidase from Spodoptera frugiperda (Sfβgly) to systematically map its residue contact network and correlate effects of mutations within and outside the active site. External mutations neighbouring the functional residues involved in catalysis and glycone-binding are deleterious, whereas mutations neighbouring the aglycone-binding site are less detrimental or even beneficial. The large dataset of new and previously characterized Sfβgly mutants supports that external perturbations are coherently transmitted to active site residues possibly through contacts and specifically disturb functional regions they interact to, reproducing the effects observed for direct mutations of functional residues. This allowed us to suggest that positions related to the aglycone-binding site are preferential targets for introduction of mutations aiming to further improve the hydrolytic activity of β-glycosidases.