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PubMed1996

X-ray structure of an anti-fungal chitosanase from streptomyces N174.

We report the 2.4 A X-ray crystal structure of a protein with chitosan endo-hydrolase activity isolated from Streptomyces N174. The structure was solved using phases acquired by SIRAS from a two-site methyl mercury derivative combined with solvent flattening and non-crystallographic two-fold symmetry averaging, and refined to an R-factor of 18.5%. The mostly alpha-helical fold reveals a structural core shared with several classes of lysozyme and barley endochitinase, in spite of a lack of shared sequence. Based on this structural similarity we postulate a putative active site, mechanism of action and mode of substrate recognition. It appears that Glu 22 acts as an acid and Asp 40 serves as a general base to activate a water molecule for an SN2 attack on the glycosidic bond. A series of amino-acid side chains and backbone carbonyl groups may bind the polycationic chitosan substrate in a deep electronegative binding cleft.

Binding Sites↗
PubMed1977

Activities of acid hydrolases in fibroblasts from normal and cystinotic children.

A study of the lysosomal hydrolases bete-galactosidase, beta-glucuronidase, N-acetyl-beta-D-glucosaminidase, and arylsulphatases A and B has been carried out on fibroblasts cultured from seven patients with cystinosis and eight control subjects. beta-Galactosidase activity was found to be consistently lower in cells derived from cystinotics, while the other enzymes studied showed no significant differences between normals and cystinotics.

Arylsulfatases↗
PubMed1996

Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core.

Barley chitinase, bacterial chitosanase, and lysozymes from goose (GEWL), phage (T4L) and hen (HEWL) all hydrolyse related polysaccharides. The proteins share no significant amino-acid similarities, but have a structurally invariant core consisting of two helices and a three-stranded beta-sheet which form the substrate-binding and catalytic cleft. These enzymes represent a superfamily of hydrolases which are likely to have arisen by divergent evolution. Based on structural criteria, we divide the hydrolase superfamily into a bacterial family (chitosanase and T4L) and a eucaryotic family represented by chitinase and GEWL. Both families contain the core but have differing N- and C-terminal domains. Inclusion of chitinase and chitosanase in the superfamily suggests the archetypal catalytic mechanism of the group is an inverting mechanism. The retaining mechanism of HEWL is unusual.

Amino Acid Sequence↗
PubMed1998

Two genes of the anaerobic fungus Orpinomyces sp. strain PC-2 encoding cellulases with endoglucanase activities may have arisen by gene duplication.

A cDNA designated celE cloned from Orpinomyces PC-2 consisted of an open reading frame encoding a polypeptide (CelE) of 477 amino acids. CelE was highly homologous to CelBs of Orpinomyces (72.3% identity) and neocallimastix (67.9% identity) and like them it had a non-catalytic repeated peptide domain (NCRPD) at the C-terminal end. The catalytic domain of CelE was homologous to glycosyl hydrolases of Family 5, found in several anaerobic bacteria. The gene of celE was devoid of introns. The recombinant proteins CelE and CelB of Orpinomyces PC-2 randomly hydrolyzed carboxymethylcellulose and cello-oligosaccharides in the pattern of endoglucanases. The results indicated that a gene of bacterial origin was duplicated to form celE and celB of Orpinomyces PC-2.

Amino Acid Sequence↗
PubMed1975

Placental acid hydrolase purification on concanavalin A-sepharose.

Crude human placental extract was chromatographed on concanavalin A-Sepharose. Using a single procedure, 20- to over 300-fold purification of six acid glycohydrolases was achieved, in most cases with a good yield. Improved yield of individual enzymes could be achieved by modifications of the method of elution. The capacity of concanavalin A-Sepharose to bind glycohydrolases from placenta was very large, and will provice a useful procedure for the large-scale purification of the human enzymes.

Chromatography↗
PubMed1996

Study on ultrastructural cytochemistry and pathogenic mechanism of Trichomonas vaginalis.

OBJECTIVE: To study the relation among enzymic ultrastructural localizations, cell organelles and functions of Trichomonas vaginalis (T. vaginalis) as well as its pathogenic mechanism. MATERIAL AND METHODS: The specimens were Trichomonas vaginalis cultured purely for several generations. After prefixation, several electron microscopic cytochemical reactions for marker enzymes of lysosome, Golgi body, mitochondrion and microbody as well as Ur-Pb-Cu impregnation were undertaken. Then the specimens were post-fixed and embedded, and the unstained ultrathin sections were observed under a transmission electron microscope. RESULTS: The activities of acid phosphatase and cytidine monophosphatase appeared in the primary and secondary lysosomes of the parasite, and the reaction product of peroxidase was found in the secondary lysosomes. It was found that lysosomes could release hydrolytic enzymes out of the cell. The reaction products of thiamine pyrophosphatase and nicotinamide adenine dinucleotide phosphatase were located within the mature-surface and intermediate saccules of Golgi body respectively. The reactions for succinate dehydrogenase, cytochrome oxidase and catalase were negative. The hydrogenosomes, endoplasmic reticulum and Golgi bodies of the parasite were densely stained by Ur-Pb-Cu impregnation method. CONCLUSIONS: T.vaginalis has lysosomal system which can release hydrolases out of the parasite, causing damage to the vaginal and urethral epithelial cells. This may be an important pathogenic mechanism of vaginitis and urethritis caused by T.vaginalis. This protozoon has well-developed Golgi bodies and rich endoplasmic reticulum. It lacks mitochondrion and microbody, but has abundant hydrogenosomes which are energy producing organelles of anaerobic metabolism and resemble microbody in morphology and mitochondrion in some functions.

Animals↗
PubMed1977

Effect of neuraminidase on the chromatographic behaviour of eleven acid hydrolases from human liver and plasma.

1. The elution profiles of eleven acid hydrolases from human liver and plasma were directly compared using a system whereby a single salt gradient was simultaneously applied to two DEAE-cellulose chromatographic columns. 2. Plasma alpha-L-fucosidase, alpha-mannosidase, alpha-galactosidase and alpha-glucosidase isoenzymes were eluted at higher salt concentrations than the corresponding liver isoenzymes whereasbeta-N-acetylglucosaminidase, beta-galactosidase, beta-glucosidase, exo-1,4-beta-xylosidase and alpha-L-arabinofuranosidase isoenzymes were eluted at lower salt concentrations. The elution profiles of beta-glucuronidase and acid phosphatase weremore complex. 3. After incubation with neuraminidase most plasma hydrolases were eluted at lower salt concentrations, however the elution patterns of beta-glucosidase, beta-xylosidase and acid phosphatase were not altered. 4. Preincubation with neuraminidase had no effect on the elution profiles of six liver hydrolases whereas the major isoenzymes of alpha-mannosidase, beta-galactosidase and alpha-L-arabinofuranosidase were eluted at markedly lower salt concentrations. Liver alpha-fucosidase and alpha-galactosidase were eluted at slightly lower salt concentrations afterincubation with neuraminidase. 5. The results are discussed in relation to thepathogenesis of Mucolipidosis II (I-cell disease), and the synthesis and packaging of lysosomal enzymes.

Acetylglucosaminidase↗
PubMed2001

The celA gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in Azospirillum irakense, is required for optimal growth on cellobiosides.

The CelA beta-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C(3), C(4), and C(5) oligosaccharides. The growth of a DeltacelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 beta-glucosidases appear to be functional alternatives for the GHF1 beta-glucosidases in the assimilation of beta-glucosides by other bacteria.

Amino Acid Sequence↗
PubMed1978

Fluorimetric assay of mycobacterial group-specific hydrolase enzymes.

Fluorigenic substrates prepared from 4-methylumbelliferone provide a simple, convenient method for detecting and assaying group-specific hydrolase activity in small quantities of whole mycobacteria. Physico-chemical properties of the enzymes such as pH dependence and heat stability may also be studied. A technique is described for studying glycosidase, hexosaminidase, acid phosphatase, and acyl esterase activity in seven mycobacterial species. Such techniques will be a useful aid to the taxonomy, identification, and quantification of mycobacteria, especially slowly growing strains in which biochemical properties are very difficult to detect by other techniques.

Acid Phosphatase↗
PubMedDate not supplied

Influence of antibody on the structure of glucans.

Glucosyltransferase (GTF) plays an essential role in the formation of the biofilm known as dental plaque and in the pathogenesis of dental caries. Mutans streptococci produce at least three distinct GTFs (GtfB, C and D), each of which forms a glucan polymer from sucrose. Glucan is a major constituent of plaque biofilm. GTF adsorbed to a surface forms glucans that differ in structure from those formed by the same enzyme in solution. In the present study, activities of GtfB and GtfC in solution or adsorbed on a surface were inhibited in the presence of a polyclonal antiserum (DS-1) to a mixture of GTFs and by immunoglobulin G (IgG) prepared from DS-1; in contrast, enzyme activity was enhanced by normal rabbit serum (NRS) and IgG from NRS. GtfD activity on a surface was enhanced by both antiserum DS-1 and NRS, and IgG prepared from either serum; GtfD activity in solution was slightly inhibited by each of the sera. The structure of GtfB and GtfC glucans formed in the presence of antiserum differed from that of controls based on linkage analyses, and on their susceptibilities to the glucanohydrolases mutanase (alpha-1,3 hydrolase) and dextranase (alpha-1,6 hydrolase); soluble products from the enzymatic digestion also differed. The results show that the effects of antibody on enzyme activity are more complex than simple inhibition or enhancement and that the presence of antibody may influence glucan structure, which clearly could impact plaque formation. The results have implications for the formation and properties of biofilms formed in other environments.

Adsorption↗
PubMed1999

Mammalian chitinase-like proteins.

Mammals express genes coding for proteins that show significant similarity to chitinases of family 18 glycosyl hydrolases. These chitinase-like proteins have no chitinase activity due to changes in critical residues in the putative active center. One of these is oviductin, a high molecular weight glycoprotein most likely involved in fertilization and protection of the tubal epithelium owing to its mucin character. Another is YKL-40 (HCgp39) produced in association with tissue remodeling. Such proteins could have a general function in morphogenesis.

Amino Acid Sequence↗
PubMed1990

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride.

The p-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HCl) that did not affect the tryptophan fluorescence of this enzyme. The Km of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combined reversibly with 1 molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.

Binding Sites↗
PubMed1995

Processing and hydrolytic mechanism of the cgkA-encoded kappa-carrageenase of Alteromonas carrageenovora.

The cgkA gene of Alteromonas carrageenovora encodes a kappa-carrageenase with a predicted mass of 44212 Da, much larger than the 35 kDa estimated from SDS/PAGE of the protein purified from culture supernatants. Immunoblotting experiments showed the presence of a protein of 44 +/- 2 kDa in both native and recombinant bacterial intracellular extracts, suggesting that the kappa-carrageenase is produced as a preproprotein which undergoes proteolytic processing twice during secretion. To determine the exact site of C-terminal cleavage, the precise mass of the purified extracellular kappa-carrageenase was measured by electrospray-ionization/mass spectrometry and found to be 31,741 +/- 3 Da. The mature kappa-carrageenase of A. carrageenovora thus appears to be composed of 275 amino acids, from residue Ala26 to residue Asn301 of the cgkA gene product. To assess the molecular mechanism of this member of family 16 of glycosyl hydrolases, hydrolysis of neocarrahexaitol by the kappa-carrageenase was monitored by gel filtration chromatography and 13C-NMR. Results show that neocarrabiitol and beta-neocarratetraose are initially formed, demonstrating that the enzyme operates with a molecular mechanism retaining the anomeric configuration. Consistent with this result, the enzyme was also shown to be able to catalyze transglycosylation.

Bacterial Proteins↗
PubMed1992

Characteristics and cariogenicity of a fructanase-defective Streptococcus mutants strain.

Polymers of D-fructose produced by a variety of oral bacteria are believed to function as extracellular carbohydrate reserves. Degradation of these polysaccharides in plaque following exhaustion of dietary carbohydrates is thought to contribute to the extent and duration of the acid challenge to the tooth surface and thus to the initiation and progression of dental caries. Streptococcus mutans produces a fructanase, the product of the fruA gene, which is capable of degrading beta(2,6)- and beta(2,1)-linked fructans that are commonly synthesized by dental plaque microorganisms. To evaluate the role of the FruA protein in exopolysaccharide metabolism and to assess the contribution of this enzyme to the pathogenic potential of S. mutans, a fructanase-deficient strain of S. mutans was constructed. Inactivation of a cloned fruA gene was accomplished in Escherichia coli by using a mini-Mu dE transposon, and then an isogenic mutant of S. mutans UA159 was constructed by allelic exchange. Successful inactivation of fruA was confirmed through the use of biochemical assays, Western blotting (immunoblotting) with anti-recombinant FruA antisera, and Southern hybridization. The data indicated that FruA was the only fructan hydrolase produced by S. mutans UA159. Inactivation of fruA had no significant effects on glucosyltransferase or fructosyltransferase activity. In the rat caries model using animals fed a high-sucrose diet and ad libitum, there were no significant differences in the number or severity of smooth surface, sulcal, or root caries elicited by the fruA mutant and the wild-type organism.

Animals↗
PubMed2005

Tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A.

Enzymes acting on polymeric substrates are frequently classified as exo or endo, reflecting their preference for, or ignorance of, polymer chain ends. Most biotechnological applications, especially in the field of polysaccharide degradation, require either endo- or exo-acting hydrolases, or they harness the essential synergy between these two modes of action. Here, we have used genomic data in tandem with structure to modify, radically, the chain-end specificity of the Cellvibrio japonicus exo-arabinanase CjArb43A. The structure of Bacillus subtilis endo-arabinanase 43A (BsArb43A) in harness with chain-end recognition kinetics of CjArb43A directed a rational design approach that led to the conversion of the Cellvibrio enzyme from an exo to an endo mode of action. One of the exo-acting mutants, D35L/Q316A, displays similar activity to WT CjArb43A and the removal of the steric block mediated by the side chains of Gln-316 and Asp-53 at the -3 subsite confers its capacity to attack internal glycoside bonds. This study provides a template for the production of tailored industrial catalysts. The introduction of subtle changes informed by comparative 3D structural and genomic data can lead to fundamental changes in the mode of action of these enzymes.

Binding Sites↗
PubMed1987

Maturational changes in terminal glycosylation of small intestinal microvillar proteins in the rat.

Studies were performed to identify rat intestinal microvillar proteins which undergo changes in terminal glycosylation during postnatal development. Pulse-labeling with [3H]fucose or N-[3H]acetylgalactosamine showed significantly higher incorporation into purified microvillar membranes of weanling than suckling rats. In contrast, the incorporation of [3H]sialic acid after pulse-labeling with N-[3H]acetylmanosamine was higher in suckling rats. SDS-polyacrylamide gel electrophoresis revealed these developmental differences in radioactive sugar incorporation to involve mainly proteins above Mr 90,000. 125I-labeled peanut lectin autoradiography revealed an Mr greater than 330,000 binding protein in suckling rats. Neuraminidase treatment of the membranes revealed the presence of sialyl-substituted sites in this protein in suckling, weaning and weanling animals, but the unmasking of sites decreased with advancing maturation. 125I-labeled Ulex europeus I autoradiography showed marked increases in binding of this lectin to Mr 66,000, 92,000, 130,000, 150,000 and greater than 330,000 proteins from weaning to weanling periods. Similar age-related increases in soybean lectin binding to Mr 130,000-150,000, and greater than 330,000 proteins were demonstrated by affinity chromatography. The Mr values of the major lectin-binding proteins were close to those reported for several hydrolases (trehalase, alkaline phosphatase, sucrase-isomaltase and glucoamylase). Comparison of the Coomassie blue-stained electrophoretograms from each age-group against the corresponding autoradiograms of lection-binding proteins led us to conclude that, while the content of these proteins in the membrane achieve their mature levels at or before weaning, their terminal glycosylation (desialylation, fucosylation, N-acetylgalactosamination) is not fully established until later development.

Aging↗
PubMed1993

New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.

301 glycosyl hydrolases and related enzymes corresponding to 39 EC entries of the I.U.B. classification system have been classified into 35 families on the basis of amino-acid-sequence similarities [Henrissat (1991) Biochem. J. 280, 309-316]. Approximately half of the families were found to be monospecific (containing only one EC number), whereas the other half were found to be polyspecific (containing at least two EC numbers). A > 60% increase in sequence data for glycosyl hydrolases (181 additional enzymes or enzyme domains sequences have since become available) allowed us to update the classification not only by the addition of more members to already identified families, but also by the finding of ten new families. On the basis of a comparison of 482 sequences corresponding to 52 EC entries, 45 families, out of which 22 are polyspecific, can now be defined. This classification has been implemented in the SWISS-PROT protein sequence data bank.

Glycoside Hydrolases↗
PubMed2001

Defoliation induces fructan 1-exohydrolase II in Witloof chicory roots. Cloning and purification of two isoforms, fructan 1-exohydrolase IIa and fructan 1-exohydrolase IIb. Mass fingerprint of the fructan 1-exohydrolase II enzymes.

The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5'- and 3'-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

Amino Acid Sequence↗