A Study of the Mechanism of Action of Taka-Amylase A1 on Linear Oligosaccharides by Product Analysis and Computer Simulation

Suganuma, Toshihiko; Matsuno, Ryuichi; Ohnishi, Masatake; Kitano, Hiromi

doi:10.1093/oxfordjournals.jbchem.a132130

Cited by 133 publications

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“…1). Binding energies have been expressed in the past as positive or negative values (23)(24)(25), but we prefer to use positive values to indicate binding (10) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

Hrmová¹,

MacGregor²,

Biely³

et al. 1998

Journal of Biological Chemistry

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A ␤-glucosidase, designated isoenzyme ␤II, from germinated barley (Hordeum vulgare L.) hydrolyzes aryl-␤-glucosides and shares a high level of amino acid sequence similarity with ␤-glucosidases of diverse origin. It releases glucose from the non-reducing termini of cellodextrins with catalytic efficiency factors, k cat /K m , that increase approximately 9-fold as the degree of polymerization of these substrates increases from 2 to 6. Thus, the enzyme has a specificity and action pattern characteristic of both ␤-glucosidases (EC 3.2.1.21) and the polysaccharide exohydrolase, (1,4)-␤-glucan glucohydrolase (EC 3.2.1.74). At high concentrations (100 mM) of 4-nitrophenyl ␤-glucoside, ␤-glucosidase isoenzyme ␤II catalyzes glycosyl transfer reactions, which generate 4-nitrophenyl-␤-laminaribioside, -cellobioside, and -gentiobioside. Subsite mapping with cellooligosaccharides indicates that the barley ␤-glucosidase isoenzyme ␤II has six substrate-binding subsites, each of which binds an individual ␤-glucosyl residue. Amino acid residues Glu 181 and Glu 391 are identified as the probable catalytic acid and catalytic nucleophile, respectively. The enzyme is a family 1 glycoside hydrolase that is likely to adopt a (␤/␣) 8 barrel fold and in which the catalytic amino acid residues appear to be located at the bottom of a funnel-shaped pocket in the enzyme.Two ␤-glucosidases of apparent molecular mass 62,000 have been purified from extracts of germinated barley grain (1-3) and can be classified in the family 1 group of glycosyl hydrolases and related enzymes (4). The two enzymes have been designated isoenzymes ␤I and ␤II, and have isoelectric points of 8.9 and 9.0, respectively. Amino acid sequence analyses reveal a single amino acid difference in the first 50 NH 2 -terminal amino acid residues, and the complete amino acid sequence of isoenzyme ␤II has been deduced from the nucleotide sequence of a corresponding cDNA clone (2).The function of the ␤-glucosidases in the germinated barley grain has not been defined unequivocally, but will clearly be related to their substrate specificities. It has been widely assumed that ␤-glucosidases prefer substrates of the type G-O-X, where G indicates the glucosyl residue and X can either be another glycosyl residue, for which the linkage position is not crucial, or a non-glycosyl aglycone group. As a result of the capacity of many ␤-glucosidases to hydrolyze glucosides with a range of glycosyl or non-glycosyl aglycone groups, non-physiological substrates such as 4-nitrophenyl ␤-D-glucopyranoside (4-NPG) 1 have been synthesized to measure activity in convenient spectrophotometric assays; the barley enzymes have also been assayed in this way. It has further been assumed that the rate of hydrolysis of oligomeric substrates by ␤-glucosidases will remain approximately constant or decrease with increasing degree of polymerization (DP) of the substrate (5). However, the barley ␤-glucosidase isoenzyme ␤II hydrolyzes (1,4)-␤-oligoglucosides much more efficiently than it hydrolyzes the aryl-␤-gluco...

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“…1). Binding energies have been expressed in the past as positive or negative values (23)(24)(25), but we prefer to use positive values to indicate binding (10) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

Hrmová¹,

MacGregor²,

Biely³

et al. 1998

Journal of Biological Chemistry

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“…To evaluate the activity of the two xylanases against xylooligosaccharides, progress curves were carried out as described previously, and the data were used to determine k cat /K m following the equation of Matsui et al (25). The bond cleavage frequency and k cat /K m data obtained from these experiments were used to calculate the ⌬G of xylose binding at each of the subsites following the method of Suganuma et al (26).…”

Section: Methodsmentioning

confidence: 99%

The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

Pell

Taylor

Gloster

et al. 2004

Journal of Biological Chemistry

158

133

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Endo-␤-1,4-xylanases (xylanases), which cleave ␤-1,4 glycosidic bonds in the xylan backbone, are important components of the repertoire of enzymes that catalyze plant cell wall degradation. The mechanism by which these enzymes are able to hydrolyze a range of decorated xylans remains unclear. Here we reveal the threedimensional structure, determined by x-ray crystallography, and the catalytic properties of the Cellvibrio mixtus enzyme Xyn10B (CmXyn10B), the most active GH10 xylanase described to date. The crystal structure of the enzyme in complex with xylopentaose reveals that at the ؉1 subsite the xylose moiety is sandwiched between hydrophobic residues, which is likely to mediate tighter binding than in other GH10 xylanases. The crystal structure of the xylanase in complex with a range of decorated xylooligosaccharides reveals how this enzyme is able to hydrolyze substituted xylan. Solvent exposure of the O-2 groups of xylose at the ؉4, ؉3, ؉1, and ؊3 subsites may allow accommodation of the ␣-1,2-linked 4-O-methyl-D-glucuronic acid side chain in glucuronoxylan at these locations. Furthermore, the uronic acid makes hydrogen bonds and hydrophobic interactions with the enzyme at the ؉1 subsite, indicating that the sugar decorations in glucuronoxylan are targeted to this proximal aglycone binding site. Accommodation of 3-linked L-arabinofuranoside decorations is observed in the ؊2 subsite and could, most likely, be tolerated when bound to xylosides in ؊3 and ؉4. A notable feature of the binding mode of decorated substrates is the way in which the subsite specificities are tailored both to prevent the formation of "dead-end" reaction products and to facilitate synergy with the xylan degradation-accessory enzymes such as ␣-glucuronidase. The data described in this report and in the accompanying paper (Fujimoto, Z., Kaneko, S., Kuno, A., Kobayashi, H., Kusakabe, I., and Mizuno, H. (2004) J. Biol. Chem. 279, 9606 -9614) indicate that the complementarity in the binding of decorated substrates between the glycone and aglycone regions appears to be a conserved feature of GH10 xylanases.

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“…Lowering the concentration of (GalpA) n to 5±10 mm to meet the criteria is technically not feasable because of the detection limits of the HPAEC±PAD system, which preclude accurate quantitation. Thus, calculation of a subsite map by the method outlined by Suganuma et al [15] was not possible for endopolygalacturonases I, II and C. Furthermore, owing to the sensitivity limits of the HPAEC± PAD system, no accurate values for K m and V max could be obtained for (GalpA) n , n . 3.…”

Section: Subsite Mappingmentioning

confidence: 99%

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C

Benen

Kester

Visser

1999

European Journal of Biochemistry

140

104

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Endopolygalacturonases I, II and C isolated from recombinant Aspergillus niger strains were characterized with respect to pH optimum, activity on polygalacturonic acid and mode of action and kinetics on oligogalacturonates of different chain length (n = 3±7).Apparent V max values using polygalacturonate as a substrate at the pH optimum, pH 4.1, were calculated as 13.8 mkat´mg ±1 , 36.5 mkat´mg ±1 and 415 nkat´mg ±1 for endopolygalacturonases I, II and C, respectively. K m values were , 0.15 mg´mL ±1 for all three enzymes. Product progression analysis using polygalacturonate as a substrate revealed a random cleavage pattern for all three enzymes and suggested processive behavior for endopolygalacturonases I and C. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 5 or 6 for endopolygalacturonase I and endopolygalacturonase C, respectively. The bond-cleavage frequencies obtained for the hydrolysis of the oligogalacturonates were used to assess subsite maps. The maps indicate that the minimum number of subsites is seven for all three enzymes.Using pectins of various degrees of esterification, it was shown that endopolygalacturonase II is the most sensitive to the presence of methyl esters. Like endopolygalacturonase II, endopolygalacturonases I, C and E, which was also included in this part of the study, preferred the non-esterified pectate. Additional differences in substrate specificity were revealed by analysis of the reaction products of hydrolysis of a mixture of pectate lyase-generated D4,5-unsaturated oligogalacturonates of degree of polymerization 4±8. Whereas endopolygalacturonase I showed a strong preference for generating the D4,5-unsaturated dimer, with endopolygalacturonase II the D4,5-unsaturated trimer accumulated, indicating further differences in substrate specificity. For endopolygalacturonases C and E both the D4,5-unsaturated dimer and trimer were observed, although in different ratios.Keywords: Aspergillus niger; endopolygalacturonase; kinetics; processivity; subsites.Saprophytic and plant pathogenic fungi and bacteria produce a vast array of enzymes capable of degrading the complex carbohydrate structures present in the plant cell wall. The most complex carbohydrate, and one of the major constituents of the middle lamella of the plant cell wall, is pectin. Owing to its complex structure, many enzymes are involved in the complete breakdown of pectin. Among these enzymes are pectin methylesterases, pectin and rhamnogalacturonan acetylesterases, pectate, pectin and rhamnogalacturonan lyases, rhamnogalacturonan hydrolases and polygalacturonases. The polygalacturonases [poly(1,4-a-d-galacturonide) glycanohydrolase, EC 3.2.1.15] hydrolyze the a-1,4 glycosidic bonds between adjacent a-dgalacturonic acid residues and are thought to act specifically on the homogalacturonan or`smooth' part of the pectin molecule.At present numerous genes encoding both exo-and endo-acting polygalactu...

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A Study of the Mechanism of Action of Taka-Amylase A1 on Linear Oligosaccharides by Product Analysis and Computer Simulation

Cited by 133 publications

References 0 publications

Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

Substrate Binding and Catalytic Mechanism of a Barley β-d-Glucosidase/(1,4)-β-d-Glucan Exohydrolase

The Mechanisms by Which Family 10 Glycoside Hydrolases Bind Decorated Substrates

Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C

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