Purification and characterization of three beta-glycosidases from midgut of the sugar cane borer,Diatraea saccharalis

Three beta-glycosidases, named betaGly1, betaGly2 and betaGly3, 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 beta-glycosidases prefer beta-glucosides to beta-galactosides, have four subsites for glucose binding and hydrolyse glucose-glucose beta-1,3 linkages better than beta-1, 4- or beta-1,6 linkages. betaGly1 and 2 were completely purified, whereas betaGly3 was isolated with a contaminant peptide that has no activity upon beta-glycosides.By using competing substrates, it was shown that betaGly 1 and 3 have one active site, whereas betaGly2 has two, one hydrolyzing natural and the other synthetic substrates. betaGly2 is the only D. saccharalis beta-glycosidase that can efficiently hydrolyse prunasin, the glycoside remaining after glucose removal from the plant glycoside amygdalin and that liberates the cyanogenic mandelonitrile. As shown elsewhere, betaGly2 activity is reduced when D. saccharalis is reared in amygdalin containing diets. From the results, we propose that the physiological role of betaGly 1 and 3 is the digestion of oligo- and disaccharides derived from hemicelluloses and of betaGly2 is glycolipid hydrolysis.Free energy relationships showed that D. saccharalis betaGly3 and Tenebrio molitor (Coleoptera) betaGly1 have active sites that bind similarly the transition states formed with different substrates. The same is also true for the active sites of D. saccharalis betaGly1 and T. molitor betaGly2. This suggests that active sites of similar enzymes are probably homologous, displaying nearly identical bonds between active site amino acids and substrate moieties.     

Characterization of a beta-glycosidase highly active on disaccharides and of a beta-galactosidase from Tenebrio molitor midgut lumen

The midgut of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae has four beta-glycosidases. The properties of two of these enzymes (betaGly1 and betaGly2) have been described elsewhere. In this paper, the characterization of the other two glycosidases (betaGly3 and betaGly4) is described. BetaGly3 has one active site, hydrolyzes disaccharides, cellodextrins, synthetic substrates and beta-glucosides produced by plants. The enzyme is inhibited by amygdalin, cellotriose, cellotetraose and cellopentaose in high concentrations, probably due to transglycosylation. betaGly3 hydrolyzes beta 1,4-glycosidic linkages with a catalytic rate independent of the substrate polymerization degree (k(int)) of 11.9 s(-1). Its active site is formed by four subsites, where subsites +1 and -1 bind glucose residues with higher affinity than subsite +2. The main role of betaGly3 seems to be disaccharide hydrolysis. BetaGly4 is a beta-galactosidase, since it has highest activity against beta-galactosides. It can also hydrolyze fucosides, but not glucosides, and has Triton X-100 as a non-essential activator (K(a)=15 microM, pH 4.5). betaGly4 has two active sites that can hydrolyze p-nitrophenyl beta-galactoside (NPbetaGal). The one hydrolyzing NPbetaGal with more efficiency is also active against methylumbellipheryl beta-D-galactoside and lactose. The other active site hydrolyzes NPbetaFucoside and binds NPbetaGal weakly. BetaGly4 hydrolyzes hydrophobic substrates with high catalytical efficiency and is able to bind octyl-beta-thiogalactoside in its active site with high affinity. The betaGly4 physiological role is supposed to be the hydrolysis of galactolipids that are found in membranes from vegetal tissues. As the enzyme has a hydrophobic site where Triton X-100 can bind, it might be activated by membrane lipids, thus becoming fully active only at the surface of cell membranes.     

Active-site mapping of a Populus xyloglucan endo-transglycosylase with a library of xylogluco-oligosaccharides

Restructuring the network of xyloglucan (XG) and cellulose during plant cell wall morphogenesis involves the action of xyloglucan endo-transglycosylases (XETs). They cleave the XG chains and transfer the enzyme-bound XG fragment to another XG molecule, thus allowing transient loosening of the cell wall and also incorporation of nascent XG during expansion. The substrate specificity of a XET from Populus (PttXET16-34) has been analyzed by mapping the enzyme binding site with a library of xylogluco-oligosaccharides as donor substrates using a labeled heptasaccharide as acceptor. The extended binding cleft of the enzyme is composed of four negative and three positive subsites (with the catalytic residues between subsites -1 and +1). Donor binding is dominated by the higher affinity of the XXXG moiety (G=Glcbeta(1-->4) and X=Xylalpha(1-->6)Glcbeta(1-->4)) of the substrate for positive subsites, whereas negative subsites have a more relaxed specificity, able to bind (and transfer to the acceptor) a cello-oligosaccharyl moiety of hybrid substrates such as GGGGXXXG. Subsite mapping with k(cat)/K(m) values for the donor substrates showed that a GG-unit on negative and -XXG on positive subsites are the minimal requirements for activity. Subsites -2 and -3 (for backbone Glc residues) and +2' (for Xyl substitution at Glc in subsite +2) have the largest contribution to transition state stabilization. GalGXXXGXXXG (Gal=Galbeta(1-->4)) is the best donor substrate with a "blocked" nonreducing end that prevents polymerization reactions and yields a single transglycosylation product. Its kinetics have unambiguously established that the enzyme operates by a ping-pong mechanism with competitive inhibition by the acceptor.     

Functional analysis of hyperthermophilic endocellulase from Pyrococcus horikoshii by crystallographic snapshots

A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01?? (1??=0.1?nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.     

Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.     

Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates.

Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values.     

Automated docking to explore subsite binding by glycoside hydrolase family 6 cellobiohydrolases and endoglucanases

Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.     

Purification and properties of a beta-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.     

Subsite Structure and Action Mode of the α-Amylase from Thermoactinomvces vulgaris

The subsite structure of Thermoactinomyces vulgaris α-amylase was estimated from its action mode and rate parameters of hydrolysis on maltooligosaccharides. These results led to the conclusion that this α-amylase has six subsites with the catalytic site located between the third and fourth subsites from the non-reducing end side. Subsite affinities were calculated to be 0.38, 5.46, 2.72 and 0.23 kcal/mol for subsites 1, 2, 5 and 6, respectively, and the sum of the affinities of subsite 3 and 4 to be ?3.41 kcal/mol. The unique action mode of this α-amylase on various substrates was interpreted in terms of the subsite structure.     

Human salivary alpha-amylase Trp58 situated at subsite -2 is critical for enzyme activity.

The nonreducing end of the substrate-binding site of human salivary alpha-amylase contains two residues Trp58 and Trp59, which belong to beta2-alpha2 loop of the catalytic (beta/alpha)(8) barrel. While Trp59 stacks onto the substrate, the exact role of Trp58 is unknown. To investigate its role in enzyme activity the residue Trp58 was mutated to Ala, Leu or Tyr. Kinetic analysis of the wild-type and mutant enzymes was carried out with starch and oligosaccharides as substrates. All three mutants exhibited a reduction in specific activity (150-180-fold lower than the wild type) with starch as substrate. With oligosaccharides as substrates, a reduction in k(cat), an increase in K(m) and distinct differences in the cleavage pattern were observed for the mutants W58A and W58L compared with the wild type. Glucose was the smallest product generated by these two mutants in the hydrolysis oligosaccharides; in contrast, wild-type enzyme generated maltose as the smallest product. The production of glucose by W58L was confirmed from both reducing and nonreducing ends of CNP-labeled oligosaccharide substrates. The mutant W58L exhibited lower binding affinity at subsites -2, -3 and +2 and showed an increase in transglycosylation activity compared with the wild type. The lowered affinity at subsites -2 and -3 due to the mutation was also inferred from the electron density at these subsites in the structure of W58A in complex with acarbose-derived pseudooligosaccharide. Collectively, these results suggest that the residue Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amylase.     

Beta-glycosylamidine as a ligand for affinity chromatography tailored to the glycon substrate specificity of beta-glycosidases

An affinity adsorbent for beta-glycosidases has been prepared by using beta-glycosylamidine as a ligand. beta-Glucosylamidine and beta-galactosylamidine, highly potent and selective inhibitors of beta-glucosidases and beta-galactosidases, respectively, were immobilized by a novel one-pot procedure involving the addition of a beta-glycosylamine and 2-iminothiolane.HCl simultaneously to a matrix modified with maleimido groups via an appropriate spacer to give an affinity adsorbent for beta-glucosidases and beta-galactosidases, respectively. This one-pot procedure enables various beta-glycosylamidine ligands to be formed and immobilized conveniently according to the glycon substrate specificities of the enzymes. A crude enzyme extract from tea leaves (Camellia sinensis) and a beta-galactosidase from Penicillium multicolor were chromatographed directly on each affinity adsorbent to give a beta-glucosidase and a beta-galactosidase to apparent homogeneity in one step by eluting the column with glucose or by a gradient NaCl elution, respectively. The beta-glucosidase and beta-galactosidase were inhibited competitively by a soluble form of the corresponding beta-glycosylamidine ligand with an inhibition constant (K(i)) of 2.1 and 0.80 microM, respectively. Neither enzyme was bound to the adsorbent with a mismatched ligand, indicating that the binding of the glycosidases was of specific nature that corresponds to the glycon substrate specificity of the enzymes. The ease of preparation and the selective nature of the affinity adsorbent should promise a large-scale preparation of the affinity adsorbent for the purification and removal of specific glycosidases according to their glycon substrate specificities.     

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

Fructosyltransferases catalyze the transfer of a fructose unit from one sucrose/fructan to another and are engaged in the production of fructooligosaccharide/fructan. The enzymes belong to the glycoside hydrolase family 32 (GH32) with a retaining catalytic mechanism. Here we describe the crystal structures of recombinant fructosyltransferase (AjFT) from Aspergillus japonicus CB05 and its mutant D191A complexes with various donor/acceptor substrates, including sucrose, 1-kestose, nystose, and raffinose. This is the first structure of fructosyltransferase of the GH32 with a high transfructosylation activity. The structure of AjFT comprises two domains with an N-terminal catalytic domain containing a five-blade β-propeller fold linked to a C-terminal β-sandwich domain. Structures of various mutant AjFT-substrate complexes reveal complete four substrate-binding subsites (−1 to +3) in the catalytic pocket with shapes and characters distinct from those of clan GH-J enzymes. Residues Asp-60, Asp-191, and Glu-292 that are proposed for nucleophile, transition-state stabilizer, and general acid/base catalyst, respectively, govern the binding of the terminal fructose at the −1 subsite and the catalytic reaction. Mutants D60A, D191A, and E292A completely lost their activities. Residues Ile-143, Arg-190, Glu-292, Glu-318, and His-332 combine the hydrophobic Phe-118 and Tyr-369 to define the +1 subsite for its preference of fructosyl and glucosyl moieties. Ile-143 and Gln-327 define the +2 subsite for raffinose, whereas Tyr-404 and Glu-405 define the +2 and +3 subsites for inulin-type substrates with higher structural flexibilities. Structural geometries of 1-kestose, nystose and raffinose are different from previous data. All results shed light on the catalytic mechanism and substrate recognition of AjFT and other clan GH-J fructosyltransferases.     

Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(-1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(-2) to Glc(-4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(-2) to Glc(-4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Subsite structure and ligand binding mechanism of glucoamylase

1. The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner. 2. The subsite structures of several amylases including glucoamylase were summarized. 3. In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration. 4. From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite 1 (nonreducing end side) where a tryptophan residue is located. 5. The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL complex. For substrates the EL may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.     

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.     

Alteration of bond-cleavage pattern in the hydrolysis catalyzed by Saccharomycopsis alpha-amylase altered by site-directed mutagenesis.

The 210th lysine (K) residue in the Saccharomycopsis alpha-amylase (Sfamy) molecule was replaced by arginine (R) and asparagine (N) residues by site-directed mutagenesis. The influences of the replacements on the bond-cleavage pattern for several substrates were analyzed. Both mutant enzymes, K210R and K210N, cleave mainly the first glycosidic bond from the reducing end of maltotetraose (G4), while the native enzyme hydrolyzes mainly the second bond from the reducing end. We changed successfully the major cleavage point in the hydrolysis reaction of G4. The 8th subsite affinities of the K210R and K210N enzymes are calculated to be +2.52 and -0.01 kcal/mol, respectively, whereas that of the native enzyme is +3.32 kcal/mol as reported in the previous paper. These affinity values suggest that the K210 residue composes the 8th subsite, one of major subsites, and that a positively charged amino residue is necessary for the 8th subsite affinity. The K210N enzyme is found to be less active for short substrates like maltotetraose (G4) than for long substrates like amylose A (approximately G18). The reduced catalytic activity specifically for the short substrates is also attributable to the remarkable decrease in the affinity of the 8th subsite.     

Interactions of barley α-amylase isozymes with Ca2 + , substrates and proteinaceous inhibitors

α-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/α)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2?+?, different substrates, and proteinaceous inhibitors for α-amylases. Isozyme specific effects of Ca^2?+? on the 80% sequence identical barley α-amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2?+? with identical ligands. A fully hydrated fourth Ca^2?+? at the interface of the AMY2/barley α-amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2?+? occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2?+?. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered ‘sugar tongs’ site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley α-amylases are reported in this review.     

Interactions of barley α-amylase isozymes with Ca2 , substrates and proteinaceous inhibitors

-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2 +, different substrates, and proteinaceous inhibitors for -amylases. Isozyme specific effects of Ca^2 + on the 80% sequence identical barley -amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2 + with identical ligands. A fully hydrated fourth Ca^2 + at the interface of the AMY2/barley -amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2 +. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley -amylases are reported in this review.     

Analysis of the key active subsites of glycoside hydrolase 13 family members

α-Amylase, pullulanase, neopullulanase, cyclomaltodextrinase (CDase), cyclomaltodextin glucanotransferase (CGTase), etc. are some of the amylolytic enzymes that act on polysaccharides. These enzymes differ from each other with respect to substrate and linkage specificities. These enzymes have been grouped into the GH13 (GH, Glycoside Hydrolase) family in the CAZy database on the basis of similarity in amino acid sequence. Members of this family share three domains viz., A, B, and C, which have several binding subsites to accommodate monomeric units of the polysaccharide substrate. Among these subsites, −2, −1, +1, and +2 subsites are the most critical subsites for catalytic activity. In the present study, the substrate analog-, inhibitor-, or product-bound 3-D structures of 24 members of GH13 family have been analyzed to identify the features of the −2, −1, +1, and +2 subsites shared by all the members for recognition of the common substrate. It is found that neither the number nor the nature of the potential hydrogen bond-forming residues is conserved with the exception of the presence of tyrosine as a stacking residue in the −1 subsite. The relative spatial disposition of the conserved subsite residues are conserved as judged by distance matrices. The backbone of the −2, −1, +1, and +2 subsites does not undergo conformational change for the recognition of the substrate. This analysis suggests that these enzymes recognize their substrate on the basis of shape of the substrate rather than on the basis of specific interactions within the binding site.     

Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.