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At least 253 records · Page 14Linked to original sources
PubMed2005

Structural insights into the beta-xylosidase from Trichoderma reesei obtained by synchrotron small-angle X-ray scattering and circular dichroism spectroscopy.

The enzyme beta-xylosidase from Trichoderma reesei, a member of glycosil hydrolase family 3 (GH3), is a glycoside hydrolase which acts at the glycosidic linkages of 1,4-beta-xylooligosaccharides and that also exhibits alpha-l-arabinofuranosidase activity on 4-nitrophenyl alpha-l-arabinofuranoside. In this work, we show that the enzyme forms monomers in solution and derive the low-resolution molecular envelope of the beta-xylosidase from small-angle X-ray scattering (SAXS) data using the ab initio simulated annealing algorithm. The radius of gyration and the maximum dimension of the beta-xylosidase are 30.3 +/- 0.2 and 90 +/- 5 A, respectively. In contrast to the fold of the only two structurally characterized members of GH3, the barley beta-d-glucan exohydrolase and beta-hexosaminidase from Vibrio cholerae, which have respectively two or one distinct domains, the shape of the beta-xylosidase indicates the presence of three distinct structural modules. Domain recognition algorithms were used to show that the C-terminal part of the amino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs demonstrate that this additional domain adopts a predominantly beta conformation.

Algorithms↗
PubMed2006

Purification, functional characterization, cloning, and identification of mutants of a seed-specific arabinan hydrolase in Arabidopsis.

This work describes the purification and characterization of an enzyme that exhibits arabinan hydrolase activity in seeds of Arabidopsis thaliana. The enzyme, designated XYL3, had an apparent molecular mass of 80 kDa when purified to homogeneity, and was identified using MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) as a putative beta-D-xylosidase that belongs to family 3 of glycoside hydrolases encoded by gene At5g09730. XYL3 hydrolysed synthetic substrates such as p-nitrophenyl-alpha-L-arabinofuranoside and p-nitrophenyl-beta-D-xyloside with similar catalytic efficiency. XYL3 released L-arabinose from (1-->5)-alpha-L-arabinofuranobiose, arabinoxylan, sugar beet arabinan, and debranched arabinan. The enzyme hydrolysed both arabinosyl-substituted side group residues and terminal arabinofuranosyl residues (1-->5)-alpha-linked to the arabinan backbone. This indicates that XYL3 is able to degrade all terminal arabinosyl residues and suggests that it participates in the in-vivo hydrolysis of arabinan. Analysis of gene expression patterns by semi-quantitative RT-PCR, in-situ hybridization and a promoter-GUS fusion demonstrated that AtBX3 was specifically expressed in the seed endosperm at the globular stage of the embryo. Immunolocalization using LM6 anti-arabinan antisera found that arabinan, the XYL3 substrate, was also present in this seed tissue. T-DNA null mutants for AtBX3 were identified. The mutant plants lacked the alpha-L-arabinofuranosidase and beta-D-xylosidase activities corresponding to XYL3. Mutants showed reduced seed size and are delayed in seedling germination compared with the wild type.

Amino Acid Sequence↗
PubMed2003

Detailed dissection of a new mechanism for glycoside cleavage: alpha-1,4-glucan lyase.

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124, 4948-4949). The classical bell-shaped pH dependence of k(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Brønsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 1.16 - 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.

1-Deoxynojirimycin↗
PubMed1979

Combined use of guar and acarbose in reduction of postprandial glycaemia.

Symptoms associated with carbohydrate malabsorption limit the usefulness to diabetics of a powerful glycoside-hydrolase inhibitor (acarbose) which reduces postprandial glycaemia. Addition of a low dose (50 mg) of a acarbose together with 14.5 g guar gum to a breakfast test meal taken by 8 healthy volunteers reduced the mean peak rise in blood-glucose at 30 min by 70%. Areas under the insulin and gastrointestinal-polypeptide response curves were also greatly reduced. No evidence of carbohydrate malabsorption, as assessed by measurement of breath hydrogen, was found during any of the test periods. When acarbose was taken alone, 3 of the 8 subjects had troublesome symptoms and the 30 min rise in blood-glucose was reduced by only 28%. Thus, combination of these two agents effectively reduces the rate of carbohydrate absorption without increasing side-effects and may make combined acarbose and guar acceptable in the management of some diabetics.

Adult↗
PubMed1991

Molecular cloning of genes from the rumen anaerobic fungus Neocallimastix frontalis: expression during hydrolase induction.

Glycoside and polysaccharide hydrolase production by the rumen anaerobic fungus, Neocalimastix frontalis is induced by the presence of crystalline cellulose. A differential screening of a cDNA library was used to isolate DNA sequences transcribed at high levels under growth conditions which induce enzyme production. Seven clones were isolated that preferentially hybridized to the induced cDNA probe versus the non-induced cDNA probe. Southern analysis showed that a cDNA clone (118) hybridized to a DNA probe encoding part of the exo-cellobiohydrolase I (CBH I) gene of Trichoderma reesei. Northern analysis demonstrated that the cDNA 118 was transcribed to yield a 2.1 kb RNA. This transcript was induced in the presence of cellulose.

Anaerobiosis↗
PubMed2006

Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins.

The white-rot basidiomycete Phanerochaete chrysosporium employs extracellular enzymes to completely degrade the major polymers of wood: cellulose, hemicellulose, and lignin. Analysis of a total of 10,048 v2.1 gene models predicts 769 secreted proteins, a substantial increase over the 268 models identified in the earlier database (v1.0). Within the v2.1 'computational secretome,' 43% showed no significant similarity to known proteins, but were structurally related to other hypothetical protein sequences. In contrast, 53% showed significant similarity to known protein sequences including 87 models assigned to 33 glycoside hydrolase families and 52 sequences distributed among 13 peptidase families. When grown under standard ligninolytic conditions, peptides corresponding to 11 peptidase genes were identified in culture filtrates by mass spectrometry (LS-MS/MS). Five peptidases were members of a large family of aspartyl proteases, many of which were localized to gene clusters. Consistent with a role in dephosphorylation of lignin peroxidase, a mannose-6-phosphatase (M6Pase) was also identified in carbon-starved cultures. Beyond proteases and M6Pase, 28 specific gene products were identified including several representatives of gene families. These included 4 lignin peroxidases, 3 lipases, 2 carboxylesterases, and 8 glycosyl hydrolases. The results underscore the rich genetic diversity and complexity of P. chrysosporium's extracellular enzyme systems.

Carboxylic Ester Hydrolases↗
PubMed2004

Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

BACKGROUND: Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. MATERIAL/METHODS: All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. RESULTS: All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. CONCLUSIONS: Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.

Adult↗