Sandro R. Marana

Molecular basis of substrate specificity in family 1 glycoside hydrolases

β‐glycosidases are active upon a large range of substrates. Besides this, subtle changes in the substrate structure may result in large modifications on the β‐glycosidase activity. The characterization of the molecular basis of β‐glycosidases substrate preference may contribute to the comprehension of the enzymatic specificity, a fundamental property of biological systems. β‐glycosidases specificity for the monosaccharide of the substrate nonreducing end (glycone) is controlled by a hydrogen bond network involving at least 5 active site amino acid residues and 4 substrate hydroxyls. From these residues, a glutamate, which interacts with hydroxyls 4 and 6, seems to be a key element in the determination of the preference for fucosides, glucosides and galactosides. Apart from this, interactions with the hydroxyl 2 are essential to the β‐glycosidase activity. The active site residues forming these interactions and the pattern of the hydrogen bond network are conserved among all β‐glycosidases. The region of the β‐glycosidase active site that interacts with the moiety (called aglycone) which is bound to the glycone is formed by several subsites (1 to 3). However, the majority of the non‐covalent interactions with the aglycone is concentrated in the first one, which presents a variable spatial structure and amino acid composition. This structural variability is in accordance with the high diversity of aglycones recognized by β‐glycosidases. Hydrophobic interactions and hydrogen bonds are formed with the aglycone, but the manner in which they control the β‐glycosidase specificity still remains to be determined. iubmb Life, 58: 63‐73, 2006

Amino acid residues involved in substrate binding and catalysis in an insect digestive β-glycosidase

A β-glycosidase (Mr 50 000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl β-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 (pKa1=4.9 and pKa2=7.5). Effect of pH on carbodiimide inactivation indicates that the pKa 7.5 group is a carboxyl. kcat and Km values were obtained for different p-nitrophenyl β-glycosides and Ki values were determined for a range of alkyl β-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature β-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E187 (proton donor) and E399 (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.

Purification, molecular cloning, and properties of a β-glycosidase isolated from midgut lumen of Tenebrio molitor (Coleoptera) larvae

Two beta-glycosidases (M(r) 59k) were purified from midgut contents of larvae of the yellow mealworm, Tenebrio molitor (Coleoptera: Tenebrionidae). The two enzymes (betaGly1 and betaGly2) 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 E379 (nucleophile) and E169 (proton donor) as catalytic amino acids based on sequence alignments. The enzymes share high homology and similarity with other insect, mammalian and plant beta-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 beta-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 beta-1,4-glucosidic bond is 21.2 s(-1) x M(-1). The putative physiological role of these enzymes is the digestion of di- and oligosaccharides derived from hemicelluloses.

Purification and properties of a β-glycosidase purified from midgut cells of Spodoptera frugiperda (Lepidoptera) larvae.

Two beta-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 beta-D-glucoside (NPbetaGlu) (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 beta-D-galactoside (NPbetaGal) better than NPbetaGlu, 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. The Mr 47,000 BG N-terminal sequence has high identity to plant beta-glycosidases and to mammalian lactase-phlorizin hydrolase, and contains the QIEGA motif, characteristic of the family of glycosyl hydrolases. The putative physiological role of this enzyme is the digestion of glycolipids (galactosidase site) and di- and oligosaccharides (cellobiase site) derived from hemicelluloses, thus resembling mammalian lactase-phlorizin hydrolase.

ULTRASTRUCTURE AND SECRETORY ACTIVITY OF ABRACRIS FLAVOLINEATA (ORTHOPTERA: ACRIDIDAE) MIDGUTS

Abstract The midgut of Abracris flavolineata adults comprises a ventriculus and six anteriorly placed caeca each displaying an anterior and a posterior lobe. Columnar cells in the caeca and anterior ventriculus present secretory vesicles originating from abundant Golgi areas, which seem to result (through exocytosis) in dark granules among the microvilli. A. flavolineata males were starved for 24 h, fed for 20 min at noon and dissected at 0, 1, 3 and 5 h after the meal. Enzyme assays were accomplished on crop and caecal contents and in subcellular fractions obtained from the isolated anterior caeca. Subcellular fractions putatively containing secretory vesicles were recognized. Digestive enzyme activity is usually low (amylase is high) in the secretory vesicles in starving insects, decreases 1 h after the meal, increases at 3 h, and thereafter decreases again (amylase remains constant). In caecal contents, digestive enzymes decrease at 1 h and increase at 3 h after the meal, the contrary being true for crop contents. Thus, in A. flavolineata caecal cells, digestive enzymes (β-glucosidase is an exception) are synthesized and secreted by exocytosis in response to feeding. Also in response to feeding, digestive enzymes are transferred from caecal contents to the crop and, after about 3 h following the meal, crop-caecal dispersed material with accompanying enzymes are translocated to the caeca, where digestion ends and absorption occurs.

Midgut β-D-glucosidases from Abracris flavolineata (Orthoptera: Acrididae). Physical properties, substrate specificities and function

Abstract A combination of gel filtration, ion-exchange chromatography, polyacrylamide gel electrophoresis, and heat inactivation data revealed the existence of three β-glucosidases with M 7 82,000 in Abracris flavolineata midgut contents: 1, a major heat-stable activity against cellobiose (cellobiase-arylβ-glucosidase); 2, a minor heat-unstable activity against p-nitrophenylβ-D-glucoside (NPβGlu) (arylβ-glucosidase); 3, an activity against octyl-β-glucoside (alkylβ-glucosidase). The cellobiase-arylβ glucosidase has a pH optimum of 5.5 and is more active on cellobiose and laminaribiose than on synthetic or natural arylβ-glucosides. Experiments involving competition between substrates and the use of inhibitors suggested that cellobiase-arylβ-glucosidase hydrolyzes cellobiose and arylβ-glucosides at different active sites. Alkylβ-glucosidase (pH optimum 4.8) has a sigmoidal activity-octyl β-glucoside-concentration profile, which changes to a hyperbolic profile in the presence of excess Triton X-100. NPβ Glu, which is hydrolyzed at the same site as octyl β-glucoside, has a hyperbolic activity-NPβGlu-concentration profile that increases in the presence of Triton X-100. It seems that amphipathic molecules activate the alkylβ-glucosidase, which is inactive on methylβ-D-glucoside and is most active on C 7 –C 10 alkyl-β glucosides. The aryl-glucosidase activity of the cellobiase-arylβ-glucosidase and the alkylβ-glucosidase are probably responsible for in vivo digestion of β1,3-glucans and glucosylceramides, respectively. Activation by detergent-like molecules is supposed to maintain high alkylβ-glucosidase activity only during plant cell membrane digestion. This avoids extensive hydrolysis of toxic plant β-glucosides which may be ingested by the insects.

Properties of digestive glycosidases and peptidases and the permeability of the peritrophic membranes of Abracris flavolineata (Orthoptera: Acrididae)

Abstract In Abracris flavolineata midguts, cellulose is hydrolyzed by at least three enzymes, whereas the most active hemicellulases are a laminarinase (pH optimum 5.7, M r 146 k) and three lichenases (pH optima in the range 5–7.3; M r values: 22, 71, and 97 k). Digestion of hemicellulose is completed by β-glucosidases described elsewhere and by an α-mannosidase (pH optimum 4.7, K m 1.7 mM). There are a major and two minor amylases (pH optimum 6.5; M r values: 42, 45, and 108 k) activated by chloride. Two of the α-glucosidases ( M r 74 and 94 k) are active on maltose and hence should finish the digestion of starch. It is not clear what is the natural substrate of the remaining α-glucosidase ( M r 93 k). The major α-galactosidase ( M r 112 k) is active on melibiose and raffinose, whereas the minor ( M r 70k) may be active on digalactosyl diglycerides. The effect of pH on azocasein hydrolysis and electrophoresis data suggest that trypsin is a major and chymotrypsin and cysteine proteinase are minor enzymes. The cysteine proteinase may derive from the leaves ingested by the grasshopper, taking into account its activity in leaves. Protein digestion is finished by two soluble aminopeptidases ( M r 92 and 105 k), a major membrane-bound aminopeptidase ( M r 97 k) and two membrane-bound dipeptidases ( M r 87 k). The sizes of the digestive enzymes recovered in A. flavolineata crops suggest that the pores of the semi-fluid caecal peritrophic membrane have diameters larger than 8.6 nm.

Single mutations outside the active site affect the substrate specificity in a β-glycosidase

A library of random mutants of the β-glycosidase Sfβgly was screened for mutations that affect its specificity for the substrate glycone (β-d-fucoside versus β-d-glucoside). Among mutations selected (T35A, R189G, Y345C, P348L, S358F, S378G, N400D, S424F, F460L, and R474H), eight occurred in the C-terminal half of Sfβgly and only two were at the active site (R189G and N400D). Tryptophan fluorescence spectra and thermal inactivation showed that the selected mutants and wild-type Sfβgly are similarly folded. Enzyme kinetics confirmed that these mutations resulted in broadening or narrowing of the preference for the substrate glycone. Structural modeling and interaction maps revealed contact pathways that connect the sites of the selected mutations through up to three interactions to the active site residues E399, W444, and E187, which are involved in substrate binding and catalysis. Interestingly, independently selected mutations (Y345C, P348L, and R189G; S424F and N400D) were placed on the same contact pathway. Moreover, (k(cat)/K(m) fucoside)/(k(cat)/K(m) glucoside) ratios showed that mutations at intermediate residues of the same contact pathway often had similar effects on substrate specificity. Finally mutations in the same contact pathway caused similar structural disturbance as evidenced by acrylamide quenching of the Sfβgly fluorescence. Based on these data, it is proposed that the effects of the selected mutations were propagated into the active site through groups of interacting residues (contact pathways) changing the Sfβgly substrate specificity.

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a β-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone‐binding site of a β‐glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site‐directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl‐type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl‐type aglycones. None of the residues investigated interacted with the terminal portion of alkyl‐type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES‡. Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.

Consumption of sugars, hemicellulose, starch, pectin and cellulose by the grasshopper Aracris flavolineata

Adults (0.61 g, fresh‐weight) of Abracris flavolineata De Geer (Orthoptera: Acrididae) feeding on Brassica oleracea acephala leaves ingest 21 mg dry‐weight/day with an approximate digestibility of 42%. Chemical determinations performed on the leaves ingested and on the feces expelled led to the determination of the approximate digestibilities (%) of the major carbohydrates of leaves as follows: soluble carbohydrates, 91; pectin, 32.1; hemicellulose, 0; starch, 66; cellulose, 15. The results are not sufficient to disregard the possibility that digestible hemicellulose polymers contaminate the pectin and the cellulose fraction. Thus, it is possible that the digestibility of hemicellulose is different from zero, and that the digestibility of pectin and cellulose are somewhat lower than reported. The data are used to propose physiological roles of the enzyme activities previously found in the A. flavolineata midgut.