4 VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland, and 5 University of Helsinki, Helsinki, Finland
Received on June 2, 2004; accepted on June 28, 2004
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Abstract |
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Key words:
(/ß)8-barrel fold
/
chitinases
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chito-oligosaccharides
/
molecular modeling
/
substrate specificity
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Introduction |
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Substrate binding as well as the catalytic mechanism have been studied for a rubber tree chitinase, hevamine, and for the two Serratia marcescens chitinases A and B. The 3D structures of these enzymes have also been solved, revealing that hevamine has an open active site architecture, while both bacterial chitinases have a more closed active site. The 3D structure of a chitinase from a pathogenic fungus, Coccioides immitis, in complex with allosamidin has also been solved recently (Bortone et al., 2002; Hollis et al., 2000
). In all cases the extended substrate-binding site allows binding of several NAG (N-acetylglucosamine, N-acetyl-ß-D-glucosamine) units (Brameld et al., 1998
; Papanikolau et al., 2001
; Terwisscha van Scheltinga et al., 1994
, 1995
; van Aalten et al., 2001
).
Carbohydrate sequences on glycoproteins, glycolipids, and proteoglycans are key ligands in different molecular recognition systems. In particular, cell-adhesion and cell-activation events triggered by carbohydrateprotein interaction are among the current topics of active research. The lactosamine Galß1-4GlcNAc and its fucosylated variant Lewis x, that is, Galß1-4(Fuc3)GlcNAc, are animal-type carbohydrates, which are important in human and mammalian biology. N-acetyllactosamine motifs function as ligands for the galectin family of lectins (Hughes, 1997
), while the fucosylated variants serve as selectin ligands (MacEver et al., 1995
; Varki, 1994
). Development of specific proteins for cleaving, synthesis, or binding analysis of those oligosaccharide structures would provide useful tools for glycobiology studies, diagnostics, and even development of glyco drugs regulating carbohydrate recognition.
We are interested in the substrate-binding specificity of glycosyl hydrolase family 18 chitinases and whether it could be engineered toward medically important oligosaccharides. We report here the purification of two fungal chitinases, Trichoderma harzianum Chit33 and Chit42, of family 18 and their enzymatic characterization using a panel of different soluble chitinous substrates and inhibitors. These two chitinases were chosen based on the fact that the smaller 33-kDa chitinase (Chit33) is homologous to the plant chitinase hevamine and is very likely to share structural similarities with this and other small plant chitinases, and the 42-kDa enzyme (Chit42) shows homology to bacterial and other fungal chitinases. We used homology modeling to build 3D structures for both fungal chitinases in complex with chito-oligosaccharides, based on the published crystal structures of family 18 enzymes. The enzyme complex structures were used to analyze the degradation patterns and establish binding models for different substrates. We are particularly interested in the specificity of the two Trichoderma chitinases toward animal-like ß-1,4-galactosylated and -1,3-fucosylated chito-oligosaccharides. This work forms a basis for understanding and modifying the substrate-binding specificity of one or both enzymes toward animal type of oligosaccharides.
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Results |
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Substrate specificity and kinetic parameters
The pH optima of both chitinases were measured at 30°C, demonstrating that the purified Chit33 has an optimal activity around pH 5 (in 50 mM NaAc buffer) on NAG3-p-nitrophenyl (PNP), and Chit42 at pH 6 (in 25 mM KPi buffer) on NAG2-4-methylumbelliferyl (MU) (data not shown). These optimum pH values were used in the all the kinetic measurements reported here. First, different-length chromophoric chito-ologosaccharides were used to characterize the degradation patterns of the two enzymes. Neither one of the chitinases showed activity against NAG-MU. Both NAG2 derivatives (NAG2-MU and NAG2-PNP) were very poor substrates for Chit33, and the enzyme seems to have a preference for longer chito-oligosaccharides (Table I). Chit42, on the other hand, is able to degrade both the NAG2-MU and NAG2-PNP efficiently. The trisaccharide derivative, NAG3-PNP, is a good substrate for both chitinases. High-performance liquid chromatography (HPLC) analysis of the hydrolysis products showed that Chit33 cleaves this substrate only at one position resulting in the formation of PNP and NAG3, whereas Chit42 cleaves NAG3-PNP at two different positions leading to the formation of NAG2, NAG-PNP, NAG3 and PNP (Table I). NAG3-MU is also degraded by both enzymes, but poor solubility of the substrate makes a detailed kinetic analysis difficult (data not shown).
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The reactivity of both enzymes for a series of galactose- and glucose-containing MU-oligosides was also determined (Table I). No hydrolysis of non-NAG-containing substrates Gal-MU, Glc-Gal-MU, and Glc2-MU was observed for either Chit33 or Chit42 (Table I).
Inhibition by transition state analogs and chito-oligosaccharides
Both enzymes were further investigated using a series of different inhibitors (Figure 1). The first inhibitor, allosamidin, is a natural pseudotrisaccharide produced by some Streptomyces species, and it is known to be a potent inhibitor of other family 18 chitinases. Additionally, two synthetic inhibitors were used, which are N-phenylcarbamate derivatives of either di-N-acetyl-chitobiose, or of N-acetyl-ß-D-glucosamine. Both inhibitors have the modification at the position C1 of the pyranose ring and they were designed to mimick a transition state of a distorted carbohydrate ring (Beer et al., 1990). The results from the inhibition studies are shown in Figure 1, where the ratio of the rate in the presence (v) and absence (v0) of the inhibitor is plotted as a function of the inhibitor concentration (Ley et al., 1997
). All experiments in one panel are performed at the same enzyme and substrate concentration, and the initial rates were measured by taking samples of the reaction mixture at four different time points. Allosamidin shows the strongest inhibitory effect both on Chit33 and Chit42, and Chit42 seems to be more strongly inhibited by allosamidin as compared to Chit33 (Figure 1). From Figure 1 it can also be seen that the two N-phenylcarbamate derivatives do not inhibit Chit33 under the experimental condition. In the case of Chit42, a small inhibitory effect is observed for the di-N-acetyl-chitobiose derivative, but the inhibition is not much stronger than with di-N-acetyl-chitobiose, the product (sugar) of this enzyme. NAG3 and NAG4, which are not cleaved by Chit33, also inhibit the enzyme, and the inhibitory effect is strongest for the longer chito-oligosaccharide NAG4.
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Figure 2 shows the different products formed from the cleavage of unmodified, galactosylated, and fucosylated NAG6 (hexa-N-acetyl-chitohexaose) by Chit33 after three different incubation times (see figure legend for details). The degradation products from the hydrolysis of NAG6 by Chit33 are shown in Figure 2 (top). NAG4 and NAG2 were the major cleavage products, and a small amount of NAG3 was also observed in all three hydrolysis experiments. NAG4 was rather resistant toward further hydrolysis by Chit33, even under the most harsh reaction conditions. In addition to the cleavage products, transglycosylation was occurring, as NAG7 was also detectable in all reaction mixtures. The second substrate, Gal-NAG6 (ß-1,4-galactosylated hexa-N-acetyl-chitohexaose), was cleaved mainly into Gal-NAG3 (and NAG3), but also some minor amounts of Gal-NAG4 could be seen (Figure 2, middle). Interestingly, Gal-NAG4 seems to be effectively cleaved further into Gal-NAG3 and NAG units by Chit33 (Figure 2, middle). Transglycosylation leading to longer oligosaccharide formation was also occurring because Gal2-NAG6 and Gal-NAG7 could be detected (Figure 2, middle). The third substrate, -1,3-fucosylated hexa-N-acetyl-chitohexaose, Fuc-NAG6 (NAGß1-4(Fuc
1-3)NAGß1-4NAG4), was cleaved effectively into Fuc-NAG5 and NAG units (Figure 2, botom). The digest contained also lots of Fuc-NAG4, but very little of NAG2, implying that the Fuc-NAG4 is a cleavage product from Fuc-NAG5. Fuc-NAG4 appeared rather stable against further hydrolysis by Chit33.
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Figure 4 shows models for binding of chito-oligosaccharide chains to T. harzianum Chit33 and Chit42, respectively. Due to conservation of most of the amino acids in the binding site, the twisted conformation of the NAG8 chain, observed in the crystal structure of S. marcescens chitinase A, also fits well into the binding site of the Chit42 model (Figure 4). Chit33 (like hevamine) seems to contain at least six and Chit42 (similar to S. marcescens chitinase A) at least seven subsites for NAG units. The enzymesubstrate complex models show that the binding groove of Chit42 contains more aromatic residues interacting with the substrate as compared to Chit33. There are five tryptophans, seven tyrosines, and one phenylalanine in the binding site of Chit42, whereas there are only one tryptophan, two tyrosines, and one phenylalanine in Chit33 (Figure 4). In Chit42 the aromatic residues are spread along the binding groove and are major determinants for formation of the subsites. In Chit33 the aromatic residues are located near the catalytic residues around subsite 1, either stacking with the oligosaccharide or stabilising the correct structure of the binding pocket.
In our Chit42 complex structure model, the hydrophobic face of the pyranose ring at the 1 site stacks with the Trp379 side chain, and Tyr44 and Phe72 form the wall in that part of the binding cleft. The residues Tyr240, Tyr44, Phe72, and Trp379 of Chit42 (corresponding to Tyr202, Tyr10, Phe41, and Trp279 of Chit33) are located in the immediate proximity of the putative catalytic amino acids and are conserved in all chitinases discussed in this paper (see Figure 4). In the subsites 3 and 2 of Chit42, most residues (Glu317, Arg53, Thr133, Glu380, Phe72, and Trp48) are conserved and are also found in the nearest homologs, chitinase A and CiX1. However, these residues are not conserved in Trichoderma Chit33 and hevamine. The sugar residue bound at the subsite 5 of Chit42 stacks against a conserved Tyr51 (Tyr170 in chitinase A and Tyr50 in CiX1), and a conserved Trp 316 flanks subsite 4 (Trp472 in chitinase A and Trp315 in CiX1). In Chit33, Ala55, Asn14, Gln13, and Asn43 participate in the formation of the binding cleft at the subsites 4 and 3 (Figure 4). Subsites +1 and +2 are lined by polar residues, Asn203, Asn204, and Gln177, found also in hevamine. Near the site where the glycosidic bond is cleaved, on one side of the binding cleft there is a very flexible loop (found both in Chit33 and hevamine), which extends from the 1 site to the +2 site and includes residues Gly249 to Gly253 of Chit33.
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Discussion |
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T. harzianum chitinases have been mostly studied because of their potential in biocontrol against other fungal pathogens and nematodes causing diseases in agricultural crops (de la Cruz et al., 1992; Hayes et al., 1994
; Limón et al., 1995
). The suggested action of chitinases there is to hydrolyze the chitin containing cell walls of the plant pathogens. We report here the enzymatic characterization of the two major chitinases of Trichoderma, Chi42 and Chi33, on various small oligosaccharides. We were particularly interested in their action on synthetic, nonnatural substrates containing galactose or fucose units. Interestingly, however, there are now reports that, for example, the fucosylated chito-oligosaccharide structure also mimics the structure of a reported nod-factor (Olsthoorn et al., 1998
). Degradation of such structures by a fungal or plant (chitinase-type) enzyme could thus be reactions found also in nature (Ovtsyna et al., 2000
).
The reasonably high homology particularly of the T. harzianum Chit 42, but also of the Chit33, with the solved 3D structures of other family 18 chitinases should allow reliable model building, especially for the ligand-binding sites in which we are interested here. The modeled structures of the two T. harzianum chitinases differ in the depth of the substrate-binding site, Chit42 having a deeper groove for chito-oligosaccharide binding. We suggest that the extra ß-sheet domain in the Chit42 model and a different tryptophan and tyrosine distribution in the substrate-binding sites are likely to be important factors that explain the different kinetic behaviour of these two enzymes in degradation studies on various chito-oligosaccharides. Figure 5 shows the subsite binding models of Chit33 and Chit42 on galactosylated and fucosylated chito-oligosaccharides based on our degradation pattern analysis (Figures 2 and 3) and the structural models (Figure 4). As can be seen from Figure 5, we suggest that Chit33 has at least six subsites (4 +2) and Chit42 at least seven subsites (5
+2). Both enzymes cleave preferentially between the second and third sugars from the reducing end of the substrate. Our kinetic data further suggest that Chit42 can degrade chitohexaose (NAG6) in a processive manner into (three) NAG2 units, and Chit33 produces NAG4 and NAG2, which are not further degraded. Apparently both enzymes have (at least) two different binding modes for chitohexaose. In the preferred binding mode NAG6 occupies the subsites 4
+2, but the chain can also bind further beyond the cleavage point, that is, to subsites 3
+3, leading to formation of two NAG3 units. A similar cleavage pattern has been observed in our earlier studies on the cellohexaose hydrolysis by Trichoderma reesei cellobiohydrolase, Cel6A (Harjunpää et al., 1996
).
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As seen from Figure 3 (middle) Chit42 can degrade animal type Gal-NAG6 (ß-1,4-galactosylated hexa-N-acetyl-chitohexaose) into Gal-NAG4 and NAG2 (or two NAG units) or into Gal-NAG3 and NAG3 units. Gal-NAG4 can also be further cleaved into Gal-NAG2 and NAG2, which are the main products after extensive hydrolysis. These data imply that Gal units can be accommodated in both subsites 4 and 3 of Chit42 (Figure 5). Chit33, on the other hand, releases from the nonreducing end of Gal-NAG6 both Gal-NAG4 and Gal-NAG3 but not Gal-NAG2 (Figure 2, middle). This implies that Gal can be accommodated only at the subsite 4 of Chit33 (Figure 5). The structural model of Chit42 reveals a conserved Thr133 residue located at subsite 3, which in the solved homologous structures is shown capable of making a hydrogen bond with the N-acetyl group of NAG (Bortone et al., 2002; Papanikolau et al., 2001
). Another conserved hydrogen bond donor, Glu317 (in Chit42), can form an additional hydrogen bond with the hydroxyl group of NAG (at position C6). On the other hand, Trp134 aligning subsite 3 stacks with the NAG ring structure but could in principle also stack against a Gal ring.
Galactosyl differs from a NAG moiety in that the hydroxyl group at C4 has an axial configuration, and it is missing the N-acetyl group at C2, thus making it smaller. Thus, on the basis of our structural model, it is plausible that a smaller Gal unit can be accommodated at subsite 3 of Chit42. At the end of the groove, at subsites 5 and 4 of Chit42, there is overall more space, and tight interactions between the protein and the saccharide units are not very likely. This probably allows more tolerance toward different substrates so that a galactose unit could be accepted (instead of a NAG unit) at both of these subsites. In the Chit33 model, subsites 3 and 4 are located on the surface of the enzyme and are more exposed when compared with the corresponding subsites in Chit42. The conserved Gln13 at subsite 3 is capable of forming a hydrogen bond with the N-acetyl group of NAG unit in subsite 3, and at subsite 4 (of Chit33), which tolerates a galactose, mainly interactions with the protein backbone are observed. Contrary to Chit42, no aromatic residues are found in Chit33 in these two subsites 3 and 4. No major contribution is expected from the axial 4-hydroxyl group in galactose, because it is proposed to point out of the binding sites in both chitinases.
The main end products of Fuc-NAG6 (-1,3-fucosylated hexa-N-acetyl-chitohexaose) hydrolysis by Chit42 are Fuc-NAG4 and NAG2 (or two NAG units) (Figure 3, bottom). In the case of Chit33, both Fuc-NAG5 and Fuc-NAG4 were produced from the nonreducing chain end (Figure 2, bottom). Fuc-NAG4 appears to be a rather stable product with both chitinases. The end product Fuc-NAG5 occupies the subsites 5
1 of Chit33, in such a way that the fucose unit is at the branch point to the NAG unit at subsite 4 (Figure 5). In the case of the shorter end product Fuc-NAG4, the fucose unit is at the branch point to the NAG unit bound to the subsite 3 in both enzymes. The conformation of an
-1,3-fucosylated saccharide has been determined in solution by nuclear magnetic resonance (NMR), showing that the branching fucose ring stacks against a NAG unit in the linear chain. It is plausible that also in the bound form the fucose unit stacks against a NAG unit occupying the subsite 3 (or 4) (Räbinä et al., 1997
).
Overall, our hydrolysis results with modified chito-oligosaccharides show that the most significant enzymesubstrate contacts are localized at subsites 1, +1, and +2 and that they are mediated by aromatic and charged residues. Our results give experimental evidence that less specific sugar interactions are found at subsites 5 2 of Chit42 and in 4
2 of Chit33, as these subsites tolerate flexibility in sugar composition. This type of flexibility in recognition should allow a good starting point for future directed evolution and protein-engineering experiments aiming at altered substrate specificity. Furthermore, the degradation pattern analysis of the chito-ologosaccharides gives some ideas for design of modified chromophoric chitinase substrates for screening purposes. We have also demonstrated that wild-type Chit42 has affinity toward a saccharide affinity column containing immobilized NAG3, whereas Chit33 elutes out already in the flow-through (data not shown). Although these data give further support to the notion that Chit42 has stronger affinity toward short chito-oligosaccharides than Chit33, they also demonstrate that this type of sugar column could be used to screen for altered substrate specificity of mutated chitinases.
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Materials and methods |
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The 33-kDa chitinase Chit33 was produced in T. harzianum strain T30 as described by Limón et al. (1995). In this strain the Chit33 can be selectively produced on minimal medium containing glucose. A 50-ml Trichoderma minimal medium + 0.2% proteose peptone preculture (2% glucose, 0.2% proteose peptone, 1.5% KH2PO4, 0.5% (NH4)2SO4, 0.6 g/L MgSO4.7H2O, 0.6 g/L CaCl2.2H2O, 5 mg/L FeSO4.7H2O, 1.6 mg/L MnSO4.H2O, 1.4 mg/L ZnSO4.7H2O, and 3.7 mg/L CoCl2.6H2O) was inoculated with the spore suspension, and the Chit33 production was checked every 24 h using western blotting (see later discussion). After 48 h of growth, 4-L Trichoderma minimal medium + 0.2% proteose peptone was inoculated with the preculture and grown for 96 h at 28°C. Glucose levels were monitored during the growth, and glucose was added when necessary. The culture supernatant was harvested by filtering the culture over a Whatman GF/B filter and frozen at 20°C.
Purification of the Chit33 and Chit42
The pH of the culture supernatant from the 42-kDa chitinase (1200 ml) was adjusted by adding 80 ml 1 M KPi, pH 7.5, and loaded onto a DEAE Sepharose FF column (Pharmacia, Uppsala, Sweden; 2.5 x 15 cm), which was equilibrated with 25 mM KPi, pH 7.5, at a flow rate of 1 ml/min. After washing the column until the A280 of the effluent reached the value of the equilibration buffer, the protein was eluted with a linear gradient (2 x 300 ml) of 01 M NaCl. Fractions were collected and Chit42 was detected by measuring the activity on MeUmb-NAG2 (see later description). The Chit42-containing fractions were pooled and concentrated, and the buffer exchanged by ultrafiltration (PES membrane with 10-kDa cut-off, Vivascience, Hanover, Germany) to 25 mM KPi, pH 6.5. The concentrated sample was further purified using a P-60 gel filtration column (BioRad, Richmond, CA; 1 x 30 cm) at a flow rate of 4 ml/h. After gel filtration, the Chit42-containing fractions were pooled and concentrated by ultrafiltration.
The purification of the Chit33 was started by concentrating the 4-L culture supernatant to 400 ml and at the same time changing the buffer to 25 mM citrate buffer, pH 3, by ultrafiltration (PES membrane with 10 kDa cut-off, Vivascience). The concentrated supernatant was loaded onto a SP-Sepharose FF column (Pharmacia; 2.5 x 10 cm) equilibrated with 25 mM citrate buffer, pH 3.0, and the column was washed with the equilibration buffer at a flow rate of 1 ml/min until the baseline was reached. The column was eluted with a linear gradient (2 x 200 ml) of 00.5 M NaCl. Fractions were collected and screened for the presence of Chit33 by western blot analysis (see later description). The Chit33-containing fractions were pooled and concentrated, and the buffer exchanged by ultrafiltration to 25 mM KPi, pH 6.5, and the sample loaded onto a Biogel P-60 gel filtration column (BioRad; 1 x 30 cm) at a flow rate of 4 ml/h. After gel filtration the Chit33-containing fractions were pooled and concentrated by ultrafiltration.
Protein analysis
Protein concentrations after purification were determined by the BCA method (Pierce, Rockford, IL) using bovine serum albumin as the standard (Smith et al., 1985). The purity of the enzymes was checked on SDSPAGE gels stained with Coomassie brilliant blue (Laemmli, 1970
). For western blot analysis rabbit polyclonal antibodies were used to detect Chit33 or Chit42 (Towbin et al., 1979
). The N-terminal amino sequences of the purified protein preparations were determined by Edman degradation using an Applied Biosystems (Foster City, CA) 949A Procise protein sequencer (Hewick et al., 1981
).
Kinetic and inhibition studies using chromophoric and fluorogenic chito-oligosaccharides
Soluble NAGn-MU (Sigma, St. Louis, MO) and NAGn-PNP (Seikagaku, Tokyo) were used for kinetic characterisation of the two enzymes. The initial rates in the presence or absence of inhibitors were determined by measuring product formation at different time points after the reaction was stopped with 0.5 M Na2CO3. All assays were performed at 30°C. The inhibition of Chit33 and Chit42 was tested with the following compounds: allosamidin, O-(chitobiosylidene) amino N-phenylcarbamate, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate, NAG2, NAG3 and NAG4. The more detailed assay conditions used in the various hydrolysis experiments are stated in the text or figure legends. For NAGn-MU cleavage, the increase in the fluorescence (i.e., the release of the MU) was measured using a Wallac Victor2 V (Turku, Finland) microtiter plate reader (excitation wavelength at 355 nm and emission wavelength at 460 nm). For NAGn-PNP cleavage, the release of the para-nitrophenol group was measured at 405 nm with a microtiter plate reader. Steady-state kinetic parameters are derived from fitting the data to the Michaelis-Menten equation using Microcal Origin 6.0 software (Northampton, MA). The enzyme concentration used in the NAGn-PNP assays was 0.51.5 µM and in the more sensitive fluorescent NAGn-MU assays 1050 nM. The detailed degradation patterns of different NAGn-PNPs were analyzed by HPLC equipped with a UV detector. The enzymatic reactions were stopped with 0.5% H3PO4 and analyzed with a Ultrahydrogel 120 column (Waters, Milford, CT) using isocratic elution with 0.1% H3PO4. GlcNAc mono- and oligomers were detected at 210 nm and free PNP groups at 300 nm.
Chemoenzymatic synthesis of ß-1,4-galactosylated and -1,3-fucosylated chito-oligosaccharides
Enzymatic synthesis of ß-1,4-galactosylated chito-oligosaccharides (Galß1-4GlcNAcß1-4R) was performed as described by Brew et al. (1968). Enzymatic synthesis of
-1,3-fucosylated chito-oligosaccharides (GlcNAcß1-4(Fuc
1-3)GlcNAcß1-4GlcNAcß1-4R) was performed as described by Natunen et al. (2001)
. Fuc-NAG6 and Fuc-NAG4 were produced by
3fucosyltransferase reaction (human recombinant Fuc-TV, Calbiochem, Darmstadt, Germany), purified by gel filtration (Superdex Peptide HR 10/30, Pharmacia) and high-pH anion exchange chromatography (CarboPac PA-1 column, Dionex, Sunnyvale, CA) separating nonreacted acceptor and characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and 1D and 2D NMR as described in Natunen et al. (1999
, 2001)
. The fucosylated oligosaccharides were at least 95% pure, Fuc-NAG6 product containing about 96% of the heptasaccharide and about 4% of NAG6 (Natunen et al., 1999
). Gal-NAG6 and Gal-NAG4 were produced in quantitative ß4galactosyltransferase reaction and purified by gel filtration (Superdex peptide 10/30, Pharmacia) and analyzed by MALDI-TOF MS showing single products without oligosaccharide impurities (Natunen et al., 1999
).
MS analysis and quantification of the hydrolysis products
MALDI-TOF MS was performed essentially as described in Räbinä et al. (1997), using the positive ion mode and 2,5-dihydroxybenzoic acid as the matrix in a BIFLEX instrument (Bruker-Franzen Analytik, Bremen, Germany). Before MS analysis, at the end of each hydrolysis reaction the oligosaccharides were purified, then the protein was denatured and together with salts removed by two-layer ion exchange chromatography using Dowex AG1-X8 (Ac, 200400 mesh, BioRad) and Dowex AG50W-X8-(H+, 200400 mesh, Biorad) (Natunen et al., 2001
; Räbinä et al., 1997
).
Binary model mixtures of chitotriose (NAG3) and chitotetraose (NAG4) were prepared from stock solutions containing 5.0 mg pure commercial glycans. Five mixtures containing 10, 25, 50, 75, and 90 mol% of NAG3 were analyzed by MALDI-TOF MS. Quite satisfactory peak height ratios (12, 24, 50, 71, and 92% for NAG3) were observed in all spectra obtained from the model mixtures. The quantities of the oligosaccharide products from the hydrolysis experiments were determined as molar percentages from the heights of the MS peaks. Our data show that the NAG3 and NAG4 peaks are at least semi-quantitatively if not quantitatively comparable to the actual amounts. It has been already shown that oligosaccharides with molecular weight of about 1000 or more give quantitative peaks in MALDI-TOF MS (Naven and Harvey, 1996; Nyman et al., 1998
). This indicates that the data for oligosaccharides of five or more units long (e.g., NAG5, NAG6, Gal-NAG4, etc.) are quantitative. The biggest inaccuracy in quantification comes from the analysis of the NAG2 and especially NAG peaks, because this region contains matrix peaks that may quench the signals, and the peaks may be also overlapped by large matrix signals. The quenching of signal may be observable in Figure 2. There should be equal amount of NAG2 and NAG4 oligosaccharides, but the amount of tetra-saccharides is about 60% whereas there are only 25% of disaccharides. The disaccharide signal is either quenched or the rest of the smaller saccharides are in the form of NAG monosaccharide. The presence of NAG2 signals thus indicates qualitative presence of the disaccharides and likely presence of substantial amount of the disaccharides.
Chitinase model building
Structural models of the T. harzianum Chit33 and Chit42 were built based on the known structures of homologous fungal and plant chitinases from family 18. Sequence comparisons and structural alignments used for model building were made using the Multalin program (Corpet, 1988) and ClustalW (Thompson et al., 1994
). The structure of chitinase-1 (CiX1) from the pathogenic fungus C. immitis (PDB-code 1D2K, Hollis et al., 2000
) sharing 53% sequence identity with Chit42 and hevamine from Hevea brasiliensis (PDB code 1LLO, Terwisscha van Scheltinga et al., 1995
) sharing 31% sequence identity with Chit33, served as template structures for model building. Chit33 and Chit42 homology models were constructed using the Insight II Homology program (Accelrys), and they were used as starting point for the construction of models in complex with hexa-N-acetyl-chitohexaose and -heptaose. The quality of the models generated was evaluated with PROCHECK (Laskowski et al., 1993
). The complex structures were created by superimposing the Chit42 model with the crystal structure of chitinase A from S. marcescens complexed with an octa-acetylchito-octaose (PDB code 1EHN, Papanikolau et al., 2001
) and the Chit33 model with the hevamine complex structures (PDB codes 1LLO and 1HVQ, Terwisscha van Scheltinga et al., 1994
, 1995
) containing allosamidin and tri-N-acetyl-chitotriose. This positioned the chito-oligosaccharides in the active sites of the T. harzianum enzymes, and further energy optimization was performed to refine the complex structures.
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Acknowledgements |
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Footnotes |
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2 Present address: Glykos Finland ltd, Viikinkaari 4, 00790 Helsinki, Finland
3 Present address: Orion Pharma, P.O. Box 65, FIN-02101 Espoo, Finland
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Abbreviations |
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References |
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