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PubMed2002

Gene cloning, sequencing, and characterization of a family 9 endoglucanase (CelA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009.

A gene encoding a beta-1,4-glucanase (CelA) belonging to subfamily E1 of family 9 of glycoside hydrolases was cloned and sequenced from the gram-positive thermoacidophile Alicyclobacillus acidocaldarius strain ATCC27009. The translated protein contains an immunoglobulin-like domain but lacks a cellulose-binding domain. The enzyme, when overproduced in Escherichia coli and purified, displayed a temperature optimum of 70 degrees C and a pH optimum of 5.5. CelA contained one zinc and two calcium atoms. Calcium and zinc are likely to be important for temperature stability. The enzyme was most active against substrates containing beta-1,4-linked glucans (lichenan and carboxy methyl cellulose), but also exhibited activity against oat spelt xylan. A striking pattern of hydrolysis on p-nitrophenyl-glycosides was observed, with highest activity on the cellobioside derivative, some on the cellotetraoside derivative, and none on the glucoside and cellotrioside derivatives. Unmodified cellooligosaccharides were also hydrolyzed by CelA. No signal peptide for transport across the cytoplasmic membrane was detected. This, together with the substrate specificity displayed, near neutral pH optimum and irreversible inactivation at low pH, suggests a role for CelA as a cytoplasmic enzyme for the degradation of imported oligosaccharides.

Acids↗
PubMed2001

Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reesei.

There are currently four proteins in family 61 of the glycoside hydrolases, from Trichoderma reesei, Agaricus bisporus, Cryptococcus neoformans and Neurospora crassa. The enzymatic activity of these proteins has not been studied thoroughly. We report here the homologous expression and purification of T. reesei Cel61A [previously named endoglucanase (EG) IV]. The enzyme was expressed in high amounts with a histidine tag on the C-terminus and purified by metal affinity chromatography. This is the first time that a histidine tag has been used as a purification aid in the T. reesei expression system. The enzyme activity was studied on a series of carbohydrate polymers. The only activity exhibited by Cel61A was an endoglucanase activity observed on substrates containing beta-1,4 glycosidic bonds, e.g. carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC) and beta-glucan. The endoglucanase activity on CMC and beta-glucan was determined by viscosity analysis, by measuring the production of reducing ends and by following the degradation of the polymer on a size exclusion chromatography system. The formation of soluble sugars by Cel61A from microcrystalline cellulose (Avicel; Merck), phosphoric acid swollen cellulose (PASC), and CMC were analysed on a HPLC system. Cel61A produced small amounts of oligosaccharides from these substrates. Furthermore, Cel61A showed activity against cellotetraose and cellopentaose. The activity of Cel61A was several orders of magnitude lower compared to Cel7B (previously EG I) of T. reesei on all substrates. One significant difference between Cel61A and Cel7B was that cellotriose was a poor substrate for Cel61A but was readily hydrolysed by Cel7B. The enzyme activity for Cel61A was further studied on a large number of carbohydrate substrates but the enzyme showed no activity towards any of these substrates.

Base Sequence↗
PubMed2001

Catalytic mechanisms and reaction intermediates along the hydrolytic pathway of a plant beta-D-glucan glucohydrolase.

BACKGROUND: Barley beta-D-glucan glucohydrolases represent family 3 glycoside hydrolases that catalyze the hydrolytic removal of nonreducing glucosyl residues from beta-D-glucans and beta-D-glucooligosaccharides. After hydrolysis is completed, glucose remains bound in the active site. RESULTS: When conduritol B epoxide and 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside are diffused into enzyme crystals, they displace the bound glucose and form covalent glycosyl-enzyme complexes through the Odelta1 of D285, which is thereby identified as the catalytic nucleophile. A nonhydrolyzable S-glycosyl analog, 4(I), 4(III), 4(V)-S-trithiocellohexaose, also diffuses into the active site, and a S-cellobioside moiety positions itself at the -1 and +1 subsites. The glycosidic S atom of the S-cellobioside moiety forms a short contact (2.75 A) with the Oepsilon2 of E491, which is likely to be the catalytic acid/base. The glucopyranosyl residues of the S-cellobioside moiety are not distorted from the low-energy 4C(1) conformation, but the glucopyranosyl ring at the +1 subsite is rotated and translated about the linkage. CONCLUSIONS: X-ray crystallography is used to define the three key intermediates during catalysis by beta-D-glucan glucohydrolase. Before a new hydrolytic event begins, the bound product (glucose) from the previous catalytic reaction is displaced by the incoming substrate, and a new enzyme-substrate complex is formed. The second stage of the hydrolytic pathway involves glycosidic bond cleavage, which proceeds through a double-displacement reaction mechanism. The crystallographic analysis of the S-cellobioside-enzyme complex with quantum mechanical modeling suggests that the complex might mimic the oxonium intermediate rather than the enzyme-substrate complex.

Catalytic Domain↗
PubMed2003

Xylosidases associated with the cell surface of Penicillium herquei IFO 4674.

Penicillium herquei IFO 4674 is a filamentous fungus that produces a large amount of hydrolases for fibrous polysaccharides. We purified two beta-xylosidases, S1 and S2. The molecular masses of S1 and S2 determined by MALDI-TOF-MS were 103,700 and 37,460 Da. The optimum pHs of S1 and S2 were 4.0 and 6.5, respectively. By several kinds of alcohols, especially glycerol, S1 was activated while S2 was unaffected or inhibited. S1 had a transxylosylation activity, while S2 did not. The s2 gene encoding xylosidase S2 was cloned by PCR with primers designed on the basis of partial amino acid sequences of S2. The s2 consisted of 1005 by encoding 335 amino acids (37,433 Da) and had no secretion signal sequence. The deduced amino acid sequence shows a high identity to that of Bacteroides ovatus xylosidase/arabinosidase (56%), which is a member of the family 43 glycoside hydrolase.

Journal Article↗
PubMed2003

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011.

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.

Acarbose↗
PubMed2004

Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase.

Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)8 barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted -1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pKa calculations and available structural information. The results indicate that the pKa of the catalytic acid (Glu144) is 'cycled' during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the -1 sugar and by increasing the pKa of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the -1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate.

Aspartic Acid↗
PubMed2001

Crystal structure of maltose phosphorylase from Lactobacillus brevis: unexpected evolutionary relationship with glucoamylases.

BACKGROUND: Maltose phosphorylase (MP) is a dimeric enzyme that catalyzes the conversion of maltose and inorganic phosphate into beta-D-glucose-1-phosphate and glucose without requiring any cofactors, such as pyridoxal phosphate. The enzyme is part of operons that are involved in maltose/malto-oligosaccharide metabolism. Maltose phosphorylases have been classified in family 65 of the glycoside hydrolases. No structure is available for any member of this family. RESULTS: We report here the 2.15 A resolution crystal structure of the MP from Lactobacillus brevis in complex with the cosubstrate phosphate. This represents the first structure of a disaccharide phosphorylase. The structure consists of an N-terminal complex beta sandwich domain, a helical linker, an (alpha/alpha)6 barrel catalytic domain, and a C-terminal beta sheet domain. The (alpha/alpha)6 barrel has an unexpected strong structural and functional analogy with the catalytic domain of glucoamylase from Aspergillus awamori. The only conserved glutamate of MP (Glu487) superposes onto the catalytic residue Glu179 of glucoamylase and likely represents the general acid catalyst. The phosphate ion is bound in a pocket facing the carboxylate of Glu487 and is ideally positioned for nucleophilic attack of the anomeric carbon atom. This site is occupied by the catalytic base carboxylate in glucoamylase. CONCLUSIONS: These observations strongly suggest that maltose phosphorylase has evolved from glucoamylase. MP has probably conserved one carboxylate group for acid catalysis and has exchanged the catalytic base for a phosphate binding pocket. The relative positions of the acid catalytic group and the bound phosphate are compatible with a direct-attack mechanism of a glycosidic bond by phosphate, in accordance with inversion of configuration at the anomeric carbon as observed for this enzyme.

Binding Sites↗
PubMed2003

Identification of the catalytic nucleophile of the family 29 alpha-L-fucosidase from Sulfolobus solfataricus via chemical rescue of an inactive mutant.

We have recently reported that a functional alpha-L-fucosidase could be expressed by a single insertional mutation in the region of overlap between the ORFs SSO11867 and SSO3060 of the hyperthermophilic Archaeon Sulfolobus solfataricus [Cobucci-Ponzano et al. J. Biol. Chem. (2003) 278, 14622-14631]. This enzyme, belonging to glycoside hydrolase family 29 (GH29), showed micromolar specificity for p-nitrophenyl-alpha-L-fucoside (pNp-Fuc) and promoted transfucosylation reactions by following a reaction mechanism in which the products retained the anomeric configuration of the substrate. The active site residues in GH29 enzymes are still unknown. We describe here the identification of the catalytic nucleophile of the reaction in the alpha-L-fucosidase from S. solfataricus by reactivation with sodium azide of the mutant Asp242Gly that shows a 10(3)-fold activity reduction on pNp-Fuc. The detailed stereochemical analysis of the fucosyl-azide produced by the mutant reactivated on pNp-Fuc revealed its inverted (beta-fucosyl azide) configuration compared with the substrate. This allows for the first time the unambiguous assignment of Asp242, and its homologous residues, as the nucleophilic catalytic residues of GH29 alpha-L-fucosidases. This is the first time that this approach is used for alpha-L-glycosidases, widening the applicability of this method.

Amino Acid Sequence↗
PubMed2004

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus.

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiADeltasp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiADeltasp were 70 degrees C and 4.5-5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70 degrees C, with half-lives of 3 h at 90 degrees C and 45 min at 95 degrees C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl2, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.

Amino Acid Sequence↗
PubMed2006

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.

Amino Acid Sequence↗
PubMed2000

Regioselectivity of enzymatic glycosylation of 6-O-acyl glycosides in supersaturated solutions.

The regioselectivity of enzymatic transglycosylation of 6-O-acetyl glycosides in supersaturated solutions was investigated using a range of commercially available enzymes, Escherichia coli, barley, and Kluyveromyces spp. beta-galactosidase, green coffee bean alpha-galactosidase, jack bean alpha-mannosidase, rice alpha-glucosidase, and almond beta-glucosidase. It has been shown that 6-O-acetyl glycosides serve as good substrates for these enzymes, which, under the reaction conditions, are "forced" to transfer monosaccharide units to the secondary hydroxyl groups of the acceptors. In a variety of transglycosylations studied the (1-3)-linked disaccharide products were the predominant regioisomers isolated. The selectivity of the reaction varied significantly depending on the acceptor glycosides and the enzyme used. Exquisite specificity was observed in some cases, but in others approximately equal quantities of two disaccharides products were isolated. In the best transfers the yield approached 30%. The methodology described offers a quick and facile route to disaccharides that may be difficult and/or time consuming to make by conventional chemical synthesis.

Carbohydrate Conformation↗
PubMed2000

Profile of glycosaminoglycan-degrading glycosidases and glycoside sulfatases secreted by human articular chondrocytes in homeostasis and inflammation.

OBJECTIVE: To determine enzymatic activities of the 8 key glycosaminoglycan-degrading glycosidases and glycoside sulfatases in cultured human articular chondrocytes and in synovial fluid from patients with osteoarthritis. METHODS: The following enzymes were analyzed: hexosaminidase and its isoenzyme A, N-acetyl-alpha-D-glucosaminidase, beta-galactosidase, beta-glucuronidase, alpha-L-iduronidase, aryl sulfatase, and galactose-6-sulfate sulfatase. Activity of the selected enzymes was analyzed by fluorometry with the aid of 4-methylumbelliferryl derivatives of the appropriate monosaccharides. RESULTS: Hexosaminidase was found to be the dominant enzyme released by chondrocytes into the extracellular compartment. Stimulation of chondrocytes with interleukin-1beta resulted in a selective increase of the extracellular hexosaminidase activity and, to a lesser degree, of the extracellular beta-galactosidase activity, without significant changes in the activity of the other studied enzymes. Analysis of the pH dependency of the enzymatic activities revealed that even at neutral pH, hexosaminidase expressed a measurable activity, much higher than the activity of the other studied enzymes. Chondrocyte apoptosis did not result in increased extracellular glycosidase activities, including hexosaminidase activity. The spectrum of glycosidase and glycoside sulfatase activities in the synovial fluid from patients with osteoarthritis was similar to that in cultured human articular chondrocytes. CONCLUSION: These data support the concept that lysosomal glycosidases, in particular hexosaminidase, represent a distinct subset of cartilage matrix-degrading enzymes that are activated by proinflammatory stimuli.

Apoptosis↗
PubMed1988

Localization and catabolism of cyanogenic glycosides.

The catabolism of cyanogenic glycosides is initiated by cleavage of the carbohydrate moiety by one or more beta-glycosidases, which yields the corresponding alpha-hydroxynitrile. Until recently, the mode by which cyanogenic disaccharides are hydrolysed was largely unclear. Investigation of highly purified beta-glycosidases from plants containing cyanogenic disaccharides has now indicated that these compounds may be degraded via two distinct pathways, depending on the plant species. beta-Glycosidases from Davallia trichomanoides and Vicia angustifolia hydrolysed (R)-vicianin and (R)-amygdalin at the aglycone-disaccharide bond producing mandelonitrile and the corresponding disaccharide. Alternatively, hydrolysis of cyanogenic disaccharides in Prunus serotina, almonds, and Linum usitatissimum involves stepwise removal of the sugar residues. The nature of these enzymes and of other beta-glycosidases responsible for hydrolysis of simple cyanogenic monosaccharides is discussed. Hydroxynitriles may decompose either spontaneously or enzymically in the presence of a hydroxynitrile lyase to produce hydrogen cyanide and an aldehyde or ketone. The major kinetic and molecular properties of hydroxynitrile lyases purified from species accumulating aromatic and aliphatic cyanogens are reviewed. Cyanogenesis occurs rapidly only after cyanogenic plant tissues are macerated, allowing glycosides access to their catabolic enzymes. The possible nature of the compartmentation which prevents cyanogenesis under normal physiological conditions is discussed in relation to our knowledge of the tissue and subcellular localizations of cyanogens and catabolic enzymes.

Aldehyde-Lyases↗